Alpha-tocopherol quinine ameliorates spatial memory deficits by reducing beta-amyloid oligomers, neuroinflammation and oxidative stress in transgenic mice with Alzheimer's disease

Accepted Manuscript Title: Alpha-tocopherol quinine ameliorates spatial memory deficits by reducing beta-amyloid oligomers, neuroinflammation and oxidative stress in transgenic mice with Alzheimer’s disease Author: Shao-wei Wang Shi-gao Yang Wen Liu Yang-xin Zhang Peng-xin Xu Teng Wang Tie-jun Ling Rui-tian Liu PII: DOI: Reference:

S0166-4328(15)30176-5 http://dx.doi.org/doi:10.1016/j.bbr.2015.09.003 BBR 9793

To appear in:

Behavioural Brain Research

Received date: Revised date: Accepted date:

1-6-2015 31-8-2015 2-9-2015

Please cite this article as: Wang Shao-wei, Yang Shi-gao, Liu Wen, Zhang Yang-xin, Xu Peng-xin, Wang Teng, Ling Tie-jun, Liu Rui-tian.Alpha-tocopherol quinine ameliorates spatial memory deficits by reducing beta-amyloid oligomers, neuroinflammation and oxidative stress in transgenic mice with Alzheimer’s disease.Behavioural Brain Research http://dx.doi.org/10.1016/j.bbr.2015.09.003 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Alpha-tocopherol quinine ameliorates spatial memory deficits by reducing betaamyloid oligomers, neuroinflammation and oxidative stress in transgenic mice with Alzheimer’s disease

Shao-wei Wang a, Shi-gao Yang a, Wen Liu

a, b

, Yang-xin Zhang a, b, Peng-xin Xu a, c, Teng Wang a,

b

, Tie-jun Ling d,*, Rui-tian Liu a,*

a

National Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese

Academy of Sciences, Beijing 100190, China b

School of Life Science, Anhui Agricultural University, Hefei 230036, China

c

School of Life Science, Ningxia University, Yinchuan 750021, China

d

State Key Laboratory of Tea Plant Biology and Utilization, Anhui Agricultural University, Hefei

230036, China

*Corresponding Author: Rui-tian Liu National Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, Haidian District, Beijing 100190, China Tel. No.: +86 10 82545017; Fax: +86 10 82545025; Email: [email protected]

Or Tie-jun Ling State Key Laboratory of Tea Plant Biology and Utilization, Anhui Agricultural University, Hefei 230036, China

Email: [email protected]

Highlights: 

Alpha-tocopherol quinine ameliorates spatial memory deficits in AD transgenic mice.



Alpha-tocopherol quinine decreases Aβ oligomer levels in the brain of AD transgenic mice.



Alpha-tocopherol quinine inhibits microglia activation by inhibiting NF-κB signaling pathway.

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Alpha-tocopherol quinine reduces cytokine production and oxidative stress in AD mice.

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Alpha-tocopherol quinine has potential therapeutic value for AD treatment.

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ABSTRACT The pathologies of Alzheimer’s disease (AD) is associated with soluble beta-amyloid (Aβ) oligomers, neuroinflammation and oxidative stress. Decreasing the levels of Aβ oligomer, glial activation and oxidative stress are potential therapeutic approaches for AD treatment. We previously found alpha-tocopherol quinine (α-TQ) inhibited Aβ aggregation and cytotoxicity, decreased the release of inflammatory cytokines and reactive oxygen species (ROS) in vitro. However, whether α-TQ ameliorates memory deficits and other neuropathologies in mice or patients with AD remains unknown. In this study, we reported that orally administered α-TQ ameliorated memory impairment in APPswe/PS1dE9 transgenic mice, decreased oxidative stress and the levels of Aβ oligomer in the brains of mice, prevented the production of inducible nitric oxide synthase and inflammatory mediators, such as interleukin-6 and interleukin-1β, and inhibited microglial activation by inhibiting NF-κB signaling pathway. These findings suggest that α-TQ has potential therapeutic value for AD treatment. Abbreviations AD: Alzheimer’s disease; Aβ, beta amyloid; α-TQ: alpha-tocopherol quinine; MWM: Morris water maze; ROS: reactive oxygen species; ThS: thioflavin S; Iba-1: ionized calcium binding adaptor molecule-1; MDA: malondialdehyde; SOD: super oxide dismutase; IL-1β: interleukin-1β; IL-6: interleukin-6 Keywords:

RRR-α-tocopherol

quinine,

Alzheimer’s

Neuroinflammation, NF-κB signaling, Oxidative stress

3

disease,

Beta-amyloid

oligomer,

4

1. Introduction Alzheimer’s disease (AD), the most common neurodegenerative disease, is characterized by accumulation of beta-amyloid (Aβ) deposits, neurofibrillary tangle formation, and progressive memory loss [1, 2]. Aβ, derived from proteolysis of amyloid precursor protein, can spontaneously self-aggregate into oligomers, protofibrils, and fibrils [3]. Soluble Aβ oligomers, but not monomers or fibrils, play a critical role in the pathogenesis of AD. The levels of Aβ oligomers in cerebrospinal fluid are related to cognitive decline in patients with AD [4]. Aβ oligomers may trigger neurotoxicity and induce inflammation responses and oxidative stress in the brains of patients with AD [3, 5, 6]. Although only part of the molecular mechanism by which Aβ oligomers exert detrimental effects has been recognized, increasing evidence suggests that agents targeting soluble Aβ oligomers may be beneficial for AD treatment [7-9]. The activated microglia and reactive astrocytes clustering around amyloid plaques are responsible for inflammation in the brains of patients with AD [10, 11]. Aβ induces glial activation, increases pro-inflammatory gene expression, and elevates secretion of cytokines, such as tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and interleukin-6 (IL-6) [12-14]. Aβ may also induce nuclear factor-κB (NF-κB) activation in glial cells and neuronal cells. NF-κB activation, which is related to inflammatory responses, was detected in the brains of patients with AD [15, 16]. Oxidative stress is implicated in the pathophysiology of AD and affected by several factors, including stimulated glia and dysfunctional mitochondria, which are major sources of highly diffusible nitric oxide radicals and oxidizing free radicals in normal aging brains and

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brains affected with AD [17]. As activated glia and NF-κB signal pathway are important in AD pathogenesis, targeting glial activity and modulating Aβ-induced NF-κB signal pathway may be feasible therapeutic strategies for the treatment of AD. RRR-α-tocopherol quinine (α-TQ) is an oxidative metabolite of α-tocopherol (α-T), which is the main component of vitamin E [18]. α-TQ exerts cellular protection against oxidative stress by reversible two-electron redox cycling [19]. It also exhibits detoxification function by removing reactive metabolites, inhibiting reactive oxygen species (ROS) through redox cycling of quinine, and producing the antioxidant hydroquinone [20]. Recent studies showed that α-TQ, which presents a therapeutic pharmacokinetic profile, was orally bioavailable and provided beneficial clinical responses in patients with Friedreich’s ataxia [21], while the low levels of tocopherols and tocotrienols in plasma increased the risk for mild cognitive impairment and AD [22]. We previously reported that α-TQ inhibited Aβ aggregation, attenuated Aβ-induced cytotoxicity in SH-SY5Y neuroblastoma cells, and decreased the release of inflammatory cytokines, and production of ROS and NO in BV-2 microglial cells in vitro [23]. However, whether α-TQ ameliorates memory deficits and other neuropathologies in mice or patients with AD remains unknown. In this study, we orally administrated α-TQ to transgenic mice with AD and determined the effects and mechanism of α-TQ on cognition and pathologies in vivo.

2. Material and Methods 2.1. Material α-TQ was extracted from Fuzhuan brick tea by using 70% aqueous acetone and purified 6

through column chromatography with silica gel (eluted with petroleum ether and acetone) and ODS

(eluted

with

MeOH).

Purity

was

analyzed

through

high-performance

liquid

chromatography. Measurement kits for Aβ40 and Aβ42 were purchased from IBL Co. Ltd. (Gunma, Japan). The antibodies used were as follows: 4G8 (monoclonal raised against Aβ17-24, Signet, MA, USA), A11 (oligomer-specific antibody, Invitrogen, Carlsbad, CA, USA), W20 (oligomer-specific single-chain variable fragment, recognizing Aβ aggregats with the molecular weight from 15 to 100 kD, developed and prepared in our laboratory[24]), OC (rabbit polyclonal raised against fibrillar oligomers, Invitrogen, Carlsbad, CA, USA), and ionized calcium-binding adaptor molecule-1 (Iba-1) polyclonal antibody (GeneTex, Alton Pkwylrvine, CA, USA). We also used p-p65, p-IκBα, and iNOS monoclonal antibody (Cell Signaling Technology, Beverly, MA, USA), as well as horseradish peroxidase-9E10 (HRP-9E10) (HRP-conjugated anti-c-myc antibody, Santa Cruz, USA) and HRP-conjugated goat anti-mouse/rabbit antibody (Gold Bridge Co., Beijing, China). Assay kits for super oxide dismutase (SOD) and malondialdehyde (MDA) measurement were obtained from Beyotime Co., Ltd. (Jiangsu, China). IL-1β and IL-6 ELISA kits were purchased from Neobioscience Technology Co., Ltd. (Beijing, China). 2.2. Animal treatment All experimental protocols were approved by the Tsinghua University (Beijing, China) Animal Care and Use Committee. Experiments were performed according to the code of practice for animal experimentation of the Animal Welfare Act and the Public Health Service Policy on Laboratory Animal Care. APPswe/PS1dE9 transgenic mice with AD expressing a chimeric mouse/human APP695, which harbors Swedish K670M/N671L mutations and human PS1 with 7

exon 9 deletion mutation, were used for spatial memory test. Littermate mice (males, 8 months of age), wild type (WT) and with AD, were given food and water ad libitum and maintained in a colony room at 22 ± 2 °C with 45% ± 10% humidity under a 12:12 h light/dark cycle. All mice were categorized into three groups: α-TQ-treated (n = 8), vehicle-treated control (n = 8), and WT (n = 8). The α-TQ-treated group was orally administered (gavage) with 100 mg/kg α-TQ daily for 4 weeks. Mice in the vehicle-treated control and WT group were treated with saline. After the last administration, the mice were trained and tested in Morris water maze (MWM). 2.3. MWM test The effect of α-TQ on the spatial cognitive performance of mice with AD was investigated through the MWM test according to a previously described method [25]. Briefly, the mice were allowed to habituate for 1 day and then tested in a water maze (1.1 m in diameter). The maze was filled with water and drained daily. The temperature of the water was maintained at 22 ± 1 °C. The platform (10 cm in diameter) was fixed 1 cm below the water surface throughout the training period, and the starting positions were counter balanced. All mice were initially assessed in the water maze to identify inherent quadrant preference and locomotor activity, and those exhibiting preference were eliminated from subsequent testing. The mice were allowed to swim for 60 s to find the platform, on which they were allowed to stay for 10 s. The mice that were unable to locate the platform were guided to it. The mice were trained twice per day for 5 consecutive days, with an inter-trial interval of 3–4 h. The swimming activity of each mouse was monitored using an overhead-mounted video camera (Sony, Tokyo, Japan) and then automatically recorded through a video tracking system. At 24 h after the last learning trial, the mice were tested in a 8

probe trial to assess their memory retention. During the probe trial test, the mice were allowed to swim for 60s in the water maze without platform. Time spent from start to first platform-site crossover (latency), time spent in the target quadrant and crossing number (number of platformsite crossovers) were determined to assess the spatial memory of mice by the tracking system. 2.4. Cerebral homogenate collection After the behavioral tests, the mice were immediately intraperitoneally anesthetized with avertin (300 mg/kg), perfused with ice-cold PBS containing heparin (10 U/mL), and then sacrificed. The brain was rapidly removed and divided. One hemisphere was rapidly dissected and homogenized. The brain tissues were bounce homogenized in radioimmunoprecipitation assay (RIPA) buffer containing protease inhibitor mixture, which consisted of 50 mM Tris (pH 7.4), 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, and 0.1% SDS. The tissues were then centrifuged at 14,000 × g for 30 min at 4 °C, and supernatant (RIPA-soluble fraction) containing soluble Aβ was collected. The pellets were suspended in guanidine buffer (5.0 M guanidine–HCl/50 mM Tris–HCl at pH 8.0) and centrifuged at 14,000 × g for 1 h at 4 °C to obtain supernatants containing insoluble Aβ (guanidine-soluble Aβ). 2.5. Measurement of Aβ40/42 RIPA-soluble and -insoluble (guanidine soluble) Aβ fractions were quantified via ELISA using Aβ40 and Aβ42 immunoassay kits according to the manufacturer’s instructions to determine the levels of Aβ in the brain. The levels of soluble and insoluble Aβ were standardized to the brain tissue weight and expressed in micrograms of Aβ per gram of brain tissue. 2.6. Dot blot 9

Mouse brain extracts were applied onto nitrocellulose membrane (Millipore, USA) to detect the levels of different Aβ forms in the brain. The membrane was blocked with 5% milk in PBST and incubated with 4G8, A11, W20, and OC antibodies at room temperature for 1 h. Finally, the bound antibodies were correspondingly probed with HRP-conjugated 9E10 or goat antimouse/rabbit antibody. Immunoreactive blots were visualized with an ECL chemiluminescence kit according to the manufacturer’s instructions. 2.7. Western blot Mouse brain extracts were loaded on 4%–12% SDS-PAGE to detect the protein levels of Iba-1, p-p65, p-IκBα, and iNOS. The separated proteins were transferred onto nitrocellulose membranes, which were then washed and incubated with primary antibodies overnight at 4 °C. The membranes were washed again and incubated with HRP-conjugated secondary antibody for 1 h at room temperature. The blots were developed with an ECL chemiluminescence kit according to the manufacturer’s instructions. The intensity of protein bands was quantified using Image J software (National Institutes of Health, USA). 2.8. Histochemistry The other hemisphere of the mouse brain was post fixed in 4% paraformaldehyde (pH 7.4) for 24 h and incubated with 30% sucrose. The brain was embedded in paraffin and sectioned (5 μm) with a Leica paraffin rotary microtome. For immunohistochemistry, the sections were subjected to deparaffination in a series of xylene and ethanol. The endogenous peroxidase in tissues was blocked with 3% H 2O2 in 80% methyl alcohol for 20 min at room temperature. Nonspecific background staining was blocked through incubation in 10% GSA and 0.3% Triton 10

X-100 at room temperature for 1 h. The tissues were incubated with primary antibodies for 1 h at room temperature, rinsed three times with PBS, added with HRP-conjugated secondary antibodies, and visualized with a 3,3′-diaminobenzidine substrate kit (Gold Bridge Co., Beijing, China). Images were collected in an Olympus BX60 microscope (Olympus Optical Co Ltd, Tokyo, Japan) by using 4/10 × objective. IpWin5 analysis software was used to qualify the microglial cells which had branches. 2.9. Measurement of SOD and MDA The activities of total SOD and Mn-SOD in the brain extracts of mice with AD were detected using SOD assay kits according to the manufacturer’s protocol. The sample was mixed with nitroblue tetrazolium and enzyme working solutions and then incubated at 37 °C for 20 min. Absorbance was determined at 560 nm. The activities of Cu/Zn-SOD were measured by subtracting Mn–SOD activity from the total SOD activity. For MDA measurement, the brain extracts were added to an Eppendorf tube containing MDA solution and then boiled for 15 min. After centrifugation at 1000 × g for 10 min, supernatant was collected in a 96-well plate. Relative MDA units were determined at 532 nm by using an MD-M5 microplate reader. 2.10. Measurement of IL-1β and IL-6 The protein levels of IL-1β and IL-6 in the cerebral homogenates were determined with IL1β and IL-6 ELISA kits according to the manufacturer’s protocols. Briefly, the brain extracts were added to a 96-well ELISA plate and then reacted with relevant primary antibodies and HRPconjugated secondary antibodies. 3,3′,5,5′-Tetramethylbenzidine (TMB) was used as substrate, 11

and absorbance was determined at 450 nm by using an MD-M5 microplate reader. 2.11. Cell culture BV-2 microglial cells were maintained in DMEM containing 10% FBS in 5% CO2 at 37 °C. Microglial cells were seeded on 24-well polystyrene plates at 50,000 cells per well. To explore the effect of α-TQ on inflammatory reaction, BV-2 cells were pretreated with or without different concentrations of α-TQ for 30 min prior to the addition of 5 μM Aβ42 to the cells. After 4 hincubation, the cells were collected and the cell lysates were centrifuged at 10,000 x g for 15 min at 4 °C. The supernatant was used for detection of NF-κB activation. 2.12. Statistical analysis Data (except MWM data) were obtained from at least three independent experiments for each condition and expressed as mean ± SD. Statistical significance was analyzed using one-way ANOVA. Statistical differences in behavioral data were determined using two-way repeated measure ANOVA.

3. Results 3.1. α-TQ treatment attenuates cognitive impairment in transgenic mice with AD After transgenic mice with AD were treated with α-TQ daily for 4 weeks, MWM behavioral tests were conducted to investigate whether the treatment ameliorates cognitive dysfunction. During acquisition training, the mice were trained to search for the hidden platform for 5 consecutive days. WT and α-TQ-treated mice with AD exhibited a remarkable decrease in escape

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latency over the 5-d training period (Fig. 1A). Although vehicle-treated mice with AD showed improved escape latency, α-TQ-treated mice with AD showed significantly shorter escape latencies than that of the control over the 5-day training period (Fig. 1A). This finding suggests that α-TQ treatment improves spatial memory in mice with AD. After the training period, the platform was removed for the probe trial. In comparison with vehicle-treated mice with AD, WT mice and α-TQ-treated mice with AD exhibited significantly shorter escape latency (the average time they spent to cross the target area for the first time) (Fig. 1B). The number of platform crossings by α-TQ-treated mice with AD was significantly higher than that of the control mice with AD (Fig. 1C). α-TQ-treated mice with AD also spent more time in the target quadrant than vehicle-treated mice with AD (Fig. 1D). Speed was not significantly different among the groups during the test session (data not shown), indicating that neither of the groups was impaired in terms of motility and exploratory activities. These results reveal that α-TQ treatment significantly attenuates the cognitive dysfunction in transgenic mice with AD. Our previous report showed that α-TQ did not apparently affect the viability and function of SH-SY5Y cells and BV2 cells in the absence of Aβ42 [23], suggesting that α-TQ may not improve memory of healthy mice.

3.2. α-TQ treatment decreases Aβ oligomer but not insoluble Aβ levels in mice with AD The total levels of soluble and insoluble Aβ in mouse brains were detected by ELISA assay kit. Compared with AD control mice, the levels of soluble rather than insoluble Aβ40 and Aβ42 in α-TQ-treated AD mice decreased, but without statistical significance (Fig. 2A and B). Soluble Aβ13

oligomer levels are related to cognitive decline in patients with AD [4]. To investigate the mechanism underlying the attenuation of cognitive deficits in mice with AD by α-TQ, we measured the levels of Aβ oligomers by using dot-blot analysis. α-TQ treatment decreased the levels of regular oligomers (W20 positive) and protofibrillar oligomers (A11 positive) by 90% and 40%, respectively, whereas the total Aβ levels (4G8 positive) and fibrillar oligomers (OC positive) in TBS-soluble fractions did not significantly change (Figs. 2C and D). Moreover, α-TQ treatment did not remarkably change the total Aβ levels (4G8 positive) and fibril levels (OC positive) in the insoluble fractions in mice with AD (Figs. 2E and F). These findings indicate that α-TQ treatment decreases the levels of some types of Aβ oligomers, except fibrils, in vivo.

3.3. α-TQ suppresses the activation of microglia in the mouse brains with AD As neuroinflammation plays a major role in the pathogenesis of AD, we examined whether α-TQ treatment would attenuate the activation of microglia in the brains of mice with AD. In comparison with WT mice, a higher amount of Iba-1-immunopositive microglia were observed in the hippocampus and cortex of mice with AD, but the amount significantly decreased after α-TQ treatment (Figs. 3A and B). To confirm these results, we assessed the Iba-1 protein levels in the mouse brains through Western-blot analysis with anti-Iba-1 antibody. Consistent with the immunohistochemistry results, Western-blot assay showed that α-TQ treatment significantly decreased Iba-1 protein levels in AD mice (Figs. 3C and D). To further confirm these results, Iba1 expression in cultured BV-2 cells was detected by adding 5μM Aβ42 with different concentrations of α-TQ. The results showed that the Iba-1 level was dose-dependently decreased 14

in 4 h-treated BV-2 cells by addition of α-TQ (Fig. 3E and F), indicating that α-TQ treatment could suppress the activation of microglia induced by Aβ in vitro and in vivo.

3.4. α-TQ attenuates microglial activation by inhibiting the NF-κB pathway The activation of NF-κB pathway stimulated by Aβ is associated with microglial activation. NF-κB subunits, namely, IκB, and p65, are phosphorylated to allow NF-κB translocate to the nucleus and activate inflammatory gene transcription. To investigate whether the inhibitory effects of α-TQ on microglia activation is caused by inhibition of the NF-κB signaling, we detected the phosphorylation of IκB-α in the brain of mice with AD. Immunohistochemistry staining results showed that α-TQ treatment decreased the levels of phosphorylated IκB-α in the hippocampus and cortex (Fig. 4A). Quantitative analysis further showed that the expression of pIκB-α significantly reduced in α-TQ-treated mice with AD compared with that in the control AD mice (Fig. 4B). Our results also showed that the phosphorylated p65 levels significantly decreased in the brains of α-TQ-treated mice with AD (Fig. 4C and D). Moreover, Aβ42 oligomers increased the phosphorylated levels of IκB-α and p65 in BV-2 cells, but α-TQ could decrease both levels in a dose-dependent manner (Fig. 4E and F). These results indicate that α-TQ may suppress Aβ-induced microglial activation by inhibiting NF-κB activation.

3.5. α-TQ reduces cytokine production and oxidative stress in vivo Activated microglial cells are significant sources of pro-inflammatory cytokines and oxidative damage [10, 11]. To detect the potential alterations in cytokine production induced by 15

α-TQ, we detected the levels of cytokines by the ELISA assay kit. The results showed that the protein levels of IL-6 and IL-1β significantly decreased in mice with AD after α-TQ treatment (Figs. 5A and B). To investigate the effect of α-TQ on ROS production in mice with AD, we further evaluated oxidative stress biomarkers, SOD and MDA. The activities of the total and Cu/Zn-SOD in vehicle-treated mice with AD significantly decreased compared with those in WT mice (Fig. 5C and D), whereas α-TQ treatment significantly increased both SOD activities. The levels of MDA, a marker of lipid peroxidation, remarkably increased in mice with AD but significantly decreased by α-TQ treatment (Fig. 5E). Excessive iNOS production is another characteristic of microglial activation. α-TQ treatment also decreased iNOS expression in the AD mice (Figs. 5F and G). These results indicate that α-TQ reduces the cytokines production, restores antioxidant activity and reduces oxidative stress in mice with AD.

4. Discussion Alpha T is the major isoform of vitamin E and the main lipophilic antioxidant in vivo. α-T can be oxidized to α-TQ and then reduced to tocopheryl hydroquinone, which is a potent radicalscavenging antioxidant in vivo [26]. α-TQ can decrease the O2− radical release in the mitochondrial respiratory chain by interacting with cytochrome complexes, and infusion of α-TQ prior to ischemia of heart could improve the recovery of hemodynamic parameters[27]. Decreasing the levels of α-T and α-TQ may change the redox balance and reduce antioxidant defenses in cervical cancer [28]. Our previous studies demonstrated that α-TQ exhibited

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neuroprotective effects in vitro, including inhibiting Aβ aggregation and cytotoxicity, disaggregating preformed fibrils, and decreasing the production of ROS and inflammatory cytokines[23]. In the present study, we revealed the effects of α-TQ on mice with AD. Recent studies on AD have focused on soluble oligomeric forms of Aβ protein, which contributes to the initial development of AD by triggering a cascade of events, including cognitive decline, neuroinflammation, and oxidative damage [29, 30]. Soluble Aβ oligomers include various species, including Aβ-derived diffusible ligands (ADDLs), Aβ*56, Aβ globulomers, and water-soluble Aβ amylo-spheroids [31]. Soluble Aβ oligomers can cause oxidative stress, inflammatory cytokine production, mitochondrial dysfunction, and cell death, thereby leading to suppression of long-term potentiation in the hippocampal slice and impaired cognitive function in vivo [32, 33]. Therefore, decreasing the levels of soluble Aβ oligomers in brains represent an optimal strategy to treat AD. Our results showed that α-TQ specifically decreased the levels of Aβ oligomers, particularly W20-positive Aβ oligomer, but not the total Aβ and insoluble Aβ in mouse brains with AD (Fig. 2). The mechanism underlying α-TQ-induced decrease of Aβ oligomers may result from the regulation of Aβ oligomers by α-TQ. Under regular conditions, such as at 37 °C and in PBS buffer, Aβ aggregates into substantial oligomers, which can be recognized by W20, rather than A11 or OC [34]. A11 bounds to oligomers formed by Aβ incubation in hexafluoro-2propanol with H2O after stirring for 24 h at 22 °C [35]. A large number of W20-recognized oligomers, rather than A11-recognized oligomers, were observed in the brains of patients or mice with AD [36]. Therefore, W20-positive oligomers may play a more important role in the neuropathology of AD and the reduction of these oligomers may contribute to the beneficial 17

effects of α-TQ on mice with AD. Microglia-induced neuroinflammation contributes to the pathogenesis of AD [37, 38]. The activated microglia around or within Aβ plaques were observed in the brains of transgenic mice and patients with AD [39, 40]. Activation of glial cells releases a wide variety of proinflammatory mediators, including IL-1β and IL-6, which potentially contribute to neuronal dysfunction and eventual death [41, 42]. Our present study revealed that α-TQ-treatment inhibited the activation of microglia, leading to the significant decrease of IL-1β and IL-6 release in the brains of AD mice. NF-κB is an important regulator of inflammation, and its activation stimulated by Aβ is associated with neuronal degeneration in the brains of patients with AD [16, 43]. NF-κB is a heterodimer composed of p50 and p65 subunits and sequestered in the cytoplasm by binding to its inhibitory subunit IκB [44]. when NF-κB stimulation, the IκB proteins are phosphorylated, ubiquitinated, and degraded, thereby allowing NF-κB to translocate to the nucleus and activate inflammatory gene transcription [45]. Thus, the effective inhibition of the NF-κB pathway may be beneficial for treatment of AD. In this study, α-TQ significantly suppressed the phosphorylation of IκB and p65 in the brains of mice with AD and in BV2 microglial cells, resulting in the decrease of activated microglia and inflammatory response in vitro and in vivo. Oxidative stress is related to microglial neuroinflammatory processes in AD pathologies, particularly in cellular and tissue damage [46]. The reduced activity of the main cellular antioxidant enzymes, such as Cu/Zn-SOD, and the increased levels of MDA, key indicator for lipid peroxidation, were observed in the brains of transgenic mice with AD [47, 48]. Hence, 18

enhancing SOD activation and reducing lipid peroxidation may be potential strategies to treat AD. In this study, gavage-administered α-TQ enhanced SOD activity and reduced MDA levels in the brains of mice with AD, suggesting that α-TQ may be an effective antioxidant for AD treatment. In conclusion, our results demonstrated that α-TQ exhibited various protective effects on mice with AD by attenuating memory impairment, decreasing Aβ oligomer levels, reducing lipid peroxidation, and restoring SOD activity. α-TQ also inhibited microglial activation and cytokine production by suppressing NF-κB signaling activation. These findings suggest that α-TQ exhibits therapeutic potential for the treatment of AD.

Competing interests The authors declare that they have no competing interests. Acknowledgments This study was supported by the National Natural Science Foundation of China (Grant No. 81171014, 31471720, 31101335) and the National Science and Technology Major Projects of New Drugs (Grant No. 2012ZX09103301-001 and 2014ZX09102045-006). References [1] Bertram L, Lill CM, Tanzi RE. The genetics of Alzheimer disease: back to the future. Neuron. 2010;68:270-81. [2] Mattson MP, Duan W, Wan R, Guo Z. Prophylactic activation of neuroprotective stress response pathways by dietary and behavioral manipulations. NeuroRx. 2004;1:111-6. [3] Querfurth HW, LaFerla FM. Alzheimer's disease. N Engl J Med. 2010;362:329-44. [4] Jongbloed W, Bruggink KA, Kester MI, Visser PJ, Scheltens P, Blankenstein MA, et al. Amyloid-beta Oligomers Relate to Cognitive Decline in Alzheimer's Disease. J Alzheimers Dis. 2014. [5] Zhou WW, Lu S, Su YJ, Xue D, Yu XL, Wang SW, et al. Decreasing oxidative stress and neuroinflammation with a multifunctional peptide rescues memory deficits in mice with Alzheimer disease.

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FIGURE LEGENDS Fig. 1. α-TQ attenuates memory deficits of transgenic mice with AD. (A) APPswe/PS1dE9 mice and WT mice treated with α-TQ or saline were given a 60 s trial twice daily to find the hidden platform. Analysis of the recorded data showed the changes in latency in searching for the hidden platform over 5 consecutive days of training. (B) Effect of α-TQ on escape latency during the memory test in the MWM probe trial without a platform. (C) Effect of α-TQ on the number of crossings (the mice crossed the position where the platform was placed during learning sessions). (D) Effect of α-TQ on the time spent in the target quadrant during the memory test in the MWM probe trial (compared with control mice with AD, *p < 0.05; **p < 0.01).

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Fig. 2. α-TQ treatment decreases Aβ oligomer but not insoluble Aβ levels in mice with AD. The amount of soluble Aβ42 and Aβ40 (A) and insoluble Aβ42 and Aβ40 (B) in cerebral homogenates were detected through sandwich ELISA using Aβ40 and Aβ42 immunoassay kits. TMB was used as the substrate, and absorbance was determined at 450 nm. Soluble Aβ in TBSsoluble fractions of the brain homogenates was detected through dot-blot assay using 4G8, W20, 24

A11, and OC antibodies (C) and qualified using IpWin5 (D). Insoluble Aβ in guanidine-soluble fractions was detected by dot-blot assay using 4G8 and OC (E) and qualified using IpWin5 (F). The results of randomly selected 3 mice representative of each group were put into C and E (compared with control mice with AD, * p < 0.05).

Fig. 3. α-TQ treatment reduces microglia activation in APP/PS1 mice. Microglia activation in the brain sections of mice with AD treated with or without α-TQ and

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control mice (WT) were visualized with anti-lba-1 antibody (A) and qualified using morphometric analysis (B). The lba-1 protein expression in mice with AD treated with or without α-TQ and WT mice was detected through western-blot assay (C) and qualified using IpWin5 (D). The results of randomly selected 3 mice representative of each group were put into C (compared with control mice with AD, *p < 0.05, ** p < 0.01). BV-2 cells were pretreated with different concentration of α-TQ for 30 min and stimulated with Aβ oligomers for 4 h. The lba-1 levels in cell lysates were detected through Western blot (E) and qualified using IpWin5 (F) (compared with BV-2 cells treating with 5μM Aβ42 alone, *p < 0.05, ** p < 0.01).

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Fig. 4. α-TQ inhibits microglial activation by inhibiting NF-κB signal pathway. The brain slices of mice with AD treated with or without α-TQ were stained with anti-p-IκB-α antibody (A), and p-IκB-α levels were qualified using IpWin5 (B). Phosphorylated p65 (p-p65) in the brains of mice with AD treated with or without α-TQ was detected through Western blot (C) and qualified by IpWin5 (D). The results of randomly selected 3 mice representative of each group were put into C (compared with control mice with AD, *p < 0.05, ** p < 0.01). BV-2 cells were pretreated with different concentration of α-TQ for 30 min and then stimulated with Aβ oligomers for 4 h. P-IκB-α and p-p65 levels in the cell lysates were detected through Western blot (E) and qualified using IpWin5 (F) (compared with BV-2 cells treating with 5μM Aβ42 alone, *p < 0.05) .

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Fig. 5. α-TQ suppresses inflammatory response and oxidative stress in brains of mice with AD. IL-6 (A) and IL-1β (B) in the cerebral homogenates of transgenic mice with AD were determined using ELISA kits. For total (C) and Cu/Zn-SOD (D) measurement, the brain extracts were mixed with NBT and enzyme-working solutions and absorbance was determined at 560 nm. For MDA

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measurement, the brain extracts were mixed with MDA detection solution, boiled, and then centrifuged. The MDA units in the supernatants were determined at 532 nm (E). iNOS in the brain extracts was detected through Western blot (F) and quantified through IpWin5 (G). The results of randomly selected 3 mice representative of each group were put into F (compared with control mice with AD, *p < 0.05, **p < 0.01).

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