Thamnolia vermicularis extract improves learning ability in APP/PS1 transgenic mice by ameliorating both Aβ and Tau pathologies

Considering the complicated pathogenesis of Alzheimer's disease (AD), multi-targets have become a focus in the discovery of drugs for treatment of this disease. In the current work, we established a multi-target strategy for discovering active reagents capable of suppressing both Aβ level and Tau hyperphosphorylation from natural products, and found that the ethanol extract of Thamnolia vermicularis (THA) was able to improve learning ability in APP/PS1 transgenic mice by inhibiting both Aβ levels and Tau hyperphosphorylation. SH-SY5Y and CHO-APP/BACE1 cells and primary astrocytes were used in cell-based assays. APP/PS1 transgenic mice [B6C3-Tg(APPswe, PS1dE9)] were administered THA (300 mg·kg−1·d−1, ig) for 100 d. After the administration was completed, the learning ability of the mice was detected using a Morris water maze (MWM) assay; immunofluorescence staining, Congo red staining and Thioflavine S staining were used to detect the senile plaques in the brains of the mice. ELISA was used to evaluate Aβ and sAPPβ contents, and Western blotting and RT-PCR were used to investigate the relevant signaling pathway regulation in response to THA treatment. In SH-SY5Y cells, THΑ (1, 10, 20 μg/mL) significantly stimulated PI3K/AKT/mTOR and AMPK/raptor/mTOR signaling-mediated autophagy in the promotion of Aβ clearance as both a PI3K inhibitor and an AMPK indirect activator, and restrained Aβ production as a suppressor against PERK/eIF2α-mediated BACE1 expression. Additionally, THA functioned as a GSK3β inhibitor with an IC50 of 1.32±0.85 μg/mL, repressing Tau hyperphosphorylation. Similar effects on Aβ accumulation and Tau hyperphosphorylation were observed in APP/PS1 transgenic mice treated with THA. Furthermore, administration of THA effectively improved the learning ability of APP/PS1 transgenic mice, and markedly reduced the number of senile plaques in their hippocampus and cortex. The results highlight the potential of the natural product THA for the treatment of AD.


Introduction
Alzheimer's disease (AD) is a progressive neurodegenerative disease that is characterized by memory loss, cognitive decline and behavior disturbance and eventually leads to death [1] . AD is characterized by a complicated pathogenesis, and the accumulation of amyloid plaques that are composed of insoluble amyloid-β (Aβ) peptides and the formation of neurofibrillary tangles (NFTs) composed of hyperphosphorylated Tau pro-teins are two typical hallmarks of this disease [2] . Accordingly, targeting Aβ or Tau phosphorylation has been long believed to be a viable strategy for drug discovery against AD.
Aβ is generated from the sequential cleavage of amyloid-β precursor protein (APP) by β-site APP cleavage enzyme 1 (BACE1) and γ-secretase [3] . In the discovery of inhibitors of Aβ production, BACE1, as a rate-limiting enzyme for Aβ generation, has received much attention. However, no BACE1 inhibitor has passed clinical trials [4] . Recently, the down-regulation of BACE1 expression was suggested as a new approach for anti-AD drug discovery [5,6] based on the fact that suppression of BACE1 expression efficiently improves AD symptoms [7,8] . Apart from Aβ inhibiting production, promoting Aβ clearance is also regarded as a valuable strategy in anti-AD research [9] . In this case, the discovery of autophagy activators has received major attention because autophagy is implicated to have an important role in Aβ clearance [10] . It has been discovered that activation of the mammalian target of rapamycin (mTOR) disrupts autophagy via phosphorylation of Unc51-like kinase 1 (ULK1), which is an initiator of the autophagy process [11] , whereas AKT (protein kinase B, PKB) as a positive regulator of mTOR is regulated by phosphoinositide 3-kinase (PI 3kinase, PI 3 K) [12] . Therefore, the PI 3 K/AKT/mTOR pathway is highly associated with autophagy-mediated Aβ clearance. Meanwhile, Tau hyperphosphorylation is believed to be the compelling cause of tauopathy and is regulated by a series of kinases and phosphatases [13] , among which glycogen synthase kinase 3β (GSK3β) and cyclin-dependent kinase (CDK5) have been identified as the main kinases for Tau pathogenesis [14,15] .
Recently, in light of the failures in clinical trials for singletarget anti-AD agents because of the complicated pathogenesis of this disease [16,17] , a multi-target-based strategy has been gradually accepted as a promising trend for the discovery of drug compounds against AD [18] . Therefore, it is expected that agents that are capable of inhibiting both Aβ levels and Tau hyperphosphorylation should be more potent anti-AD agents [19] . Accordingly, considering that natural products are major sources of bioactive agents based on their large-scale structural and acting target diversities, we have recently constructed a "one stone, two birds" platform against our laboratory's in-house natural product library to identify agents that are able to inhibit both Aβ levels and Tau hyperphosphorylation. As a result, THA, an extract of Thamnolia vermicularis, was discovered to improve learning ability in APP/PS1 transgenic mice by ameliorating Aβ/Tau pathology.
Thamnolia vermicularis mainly grows in snowy areas at altitudes 4000 m above sea level (eg, Yunnan and Sichuan Provinces of China) and is named "White snow tea" ("Baixuecha" in Chinese) in China [20] . Thamnolia vermicularis has been used in traditional Chinese medicine for many years [21] , and pharmacological research has suggested that it has antioxidant and anticancer functions and can be used for the treatment of hypertension, cough, and neurasthenia [20,22] . Recently, several phenolic compounds (eg, vermicularin, squamatic acid, barbatinic acid, D-arabitol, mannitol and baeomycesic acid) have been reported to be extracted from this type of lichen plant [23] .
In the current work, we report that the ethanol extract of Thamnolia vermicularis (THA) improved learning ability in APP/PS1 transgenic mice by inhibiting both Aβ levels and Tau hyperphosphorylation. The mechanisms underlying the THΑ-mediated pharmacological events against AD were intensively investigated. Our results highlight the potential of THA in the treatment of AD.
Preparation of THA THA was prepared according to a previously published method [24]  Thamnolia vermicularis (2.0 kg) was extracted with 95% EtOH under reflux (20 L ×3) for 2 h. The extract was filtered, and the filtration was concentrated under reduced pressure to obtain 2 L of mixed suspension. The suspension was sedimentated for 5 h, followed by centrifugation at 4000 rounds per minute to yield a residue, and then dried to yield 150 g of the total phenolic acids. According to HPLC-UV analysis, the contents of squamatic acid (1) and baeomycesic acid (2) ( Figure 1A) in the total phenolic acids were 42% and 46%, respectively.
Cell culture In our current work, spontaneous over-expression of Aβ is needed for evaluating the Aβ inhibitory activity of the compound; therefore, the high Aβ expression of CHO-APP/ BACE1 cells may facilitate the assay. According to the published reports, BV 2 microglia, a main immunocompetent cell in the CNS, functions potently in phagocytosing exogenous Aβ [25][26][27] . SH-SY5Y cells, a neuroblastoma cell line that closely resembles primary neurons, may be used instead of primary neurons [28][29][30] , while primary astrocytes play an important role in autophagy-mediated Aβ clearance in vivo and are considered more suitable for the related assays [31][32][33] . Additionally, published reports have revealed that SH-SY5Y cells, as human neuroblastoma cells, are being widely used in the study of Tau pathology [28,34,35] , and tauopathy was also discovered in glial cells and astrocytes [36,37] . Thus, we used SH-SY5Y, BV 2 and astrocyte cells to study the regulation of THA against the signaling pathways involved in Aβ/Tau pathology. In addition, we did not use primary neurons because of their relatively low expression level of Aβ and the restricted number of cells [29,32,33] . As stated above, primary astrocytes play an important role in autophagy-mediated Aβ clearance in vivo and are considered more suitable for the related assay. Primary astrocytes were thus applied to further confirm the results obtained in the SH-SY5Y cells.

ELISA assay
Aβ/sAPPβ content assay CHO-APP/BACE1 cells were incubated with different concentrations of THA (20, 10, 1, or 0 mg/L) for 24 h, and the supernatant was collected, to which a complete protease inhibitor mixture was added (Thermo, USA). The contents of Aβ and sAPPβ were measured using human Aβ 40 (Invitrogen, KHB3481, USA) and sAPPβ (Immuno-Biological Laboratories, Japan) ELISA kits, respectively.

Aβ clearance assay
An exogenous Aβ clearance assay of the cells was performed according to the published Landreth approach [38,39] . BV 2 cells, astrocytes and SH-SY5Y cells were incubated with different concentrations of THA (20, 10, 1, or 0 μg/mL) for 20 h and then treated with 2 μg/mL exogenous Aβ 40 or Aβ 42 for 3 h. Extracellular membrane exogenous Aβ was washed away three times using PBS buffer (pH 7.4), and the cells were then lysed in 50 mmol/L Tris buffer (pH 8.0) containing 5 mol/L guanidine hydrochloride. After the lysate was centrifuged at 20 000×g for 30 min at 4 °C, the Aβ 40 and Aβ 42 in the supernatant were detected using human Aβ 40 and Aβ 42 ELISA kits, respectively.
Brains were assayed according to a previously published method [40] . Hippocampal and cortical samples were homogenized in 50 mmol/L Tris buffer (pH 8.0) containing 5 mol/L guanidine hydrochloride by manual grinding, followed by lysis at 4 °C for 1 h. The homogenates were centrifuged at 20 000×g at 4 °C for 30 min, and the content of Aβ 40 or Aβ 42 in the supernatant was detected using human Aβ 40 or Aβ 42 ELISA kits, respectively.
In the assays, the total protein concentration of the sample was determined using a BCA protein assay kit (Thermo, USA), and all Aβ concentration values were normalized to the total protein concentration.

Western blotting
Cell-based assay SH-SY5Y cells, BV 2 cells, CHO-APP/BACE1 cells and astrocytes were treated with varying concentrations of THA for 24 h and lysed with RIPA buffer (Thermo, USA) containing a protease inhibitor cocktail (Thermo, USA). Protein concentration was determined using a BCA protein assay kit (Thermo, USA). The lysis solutions were mixed with 2× loading buffer (4% SDS, 62.5 mmol/L Tris-HCI, pH 6.8, 25% glycerol, and 0.1% bromophenol blue), boiled for 15 min at 99 °C, and then centrifuged at 20 000×g for 10 min.

Tissue-based assay
The cortices from four mouse brains in each group were homogenized by manual grinding in RIPA buffer (Thermo, USA) and kept on ice for 1 h to lyse completely. The homogenates were then centrifuged at 20 000×g at 4 °C for 30 min. The total protein concentration of the supernatant was determined using a BCA protein assay kit (Thermo, USA). Equal amounts of homogenates (2 mg/mL protein) were mixed with 2× SDS-PAGE sample buffers, and equal amounts of mixture (3 mg/mL protein) were then boiled for 15 min at 99 °C followed by centrifugation at 20 000×g for 10 min.

BACE1 activity assay
A BACE1 (β-secretase) activity kit (Invitrogen, USA) was used to determine the inhibition of THA against BACE1 activity in vitro. Briefly, BACE1 substrate (250 nmol/L), BACE1 enzyme (0.35 U/mL) and varied concentrations of THA were mixed gently and then incubated for 1 h at 37 °C in cells dark. The fluorescence intensity was measured at excitation and emission wavelengths of 545 and 585 nm, respectively.

PI 3 K activity assay
Inhibition of THA on PI 3 K enzymatic activity in vitro was assayed using a PI-3 kinase alpha (p110α/p85α) active assay kit (Echelon Biosciences Inc, USA) according to the manufacturer's protocol. In brief, PIP 2 substrate (10 μmol/L), PI 3 K kinase (0.36 μmol/L) and various concentrations of THA were mixed and incubated for 3 h at room temperature, followed by the addition of 90 μL of kinase stop solution to stop the reaction. Then, 60 μL of cells reaction supernatant was transferred to a clean tube, followed by the addition of PIP 3 detector, and incubated for 1 h at room temperature. The absorbance at 450 nm was read. The result indicated that the colorimetric signal was inversely proportional to the amount of PI(3,4,5)P 3 produced by PI-3 kinase.

CDK5/P25 kinase enzyme assay
The effect of THA on CDK5/P25 kinase activity was determined using a CDK5/P25 Activity Assay Kit (Promega catalog #V3231) according to the manufacturer's protocol. Briefly, a CDK5 enzyme reaction system was directly incubated with different concentrations of THA or PHA-793887 (published CDK5 inhibitor [41] ) for 10 min, and then an ADP-GloTM Kinase Assay Kit (Promega catalog #V9101) was used to measure the activity of CDK5/P25 by quantifying the amount of ADP produced during the kinase reaction.

AMPK activity assay
The effect of THA on AMPK activity was determined using an ADP Hunter Plus Assay Kit (DiscoveRX, lot #13C1816), in which AMPK activity was investigated by quantifying the amount of ADP produced during the kinase reaction. In brief, varied concentrations of THA were incubated with AMPKα2β1γ1 (500 ng/mL) for 30 min on ice, followed by the addition of ATP (100 μmol/L) and SAMS (100 μmol/L) to start the reaction in cells dark. After 30 min, an ADP Hunter Plus Assay Kit was used to test ADP content. The fluorescence intensity was measured with excitation and emission wavelengths of 530 nm and 590 nm, respectively.

Autophagy activation by mRFP-GFP adenoviral vector
The effects of THA on autophagic flux and autolysosome formation were determined by using a confocal laser scanning microscope (CLSM)-based assay (HanBio, China), according to the provided kit protocol. Briefly, SH-SY5Y cells with an expression vector encoding mRFP-GFP fluorescence-tagged LC3 (mRFP-GFP-LC3) were incubated with different concentrations of THA or rapamycin (known autophagy inhibitor [42] ) for 24 h at 37 °C. After cell fixation, the cells were imaged for green fluorescent protein (GFP) and red fluorescent protein (RFP) using an Olympus Fluoview FV1000 confocal microscope (Olympus, Japan). Autophagic flux was determined by evaluating the number of mRFP puncta (puncta/cell were counted).

Real-time PCR analysis
The total mRNA from cells and mouse brains was extracted using TRIzol Reagent according to the protocols of commercial kits. Complementary DNA synthesis was performed with oligo-dT primers according to the instructions of a reverse-PCR kit (TaKaRa Bio, Japan), and real-time PCR was carried out using SYBR Premix Ex Taq TM on a DNA Engine Opticon TM2 system (TaKaRa Bio, Japan). The primers used are listed in Table 1.

Animal experiments
All animal experiments were carried out according to the Institutional Ethical Guidelines of Shanghai Institute of Materia Medica, Chinese Academy of Sciences, on animal care. The APP/PS1 transgenic mice [B6C3-Tg (APP swe , PS1 dE9 )] were purchased from Jackson Laboratory (Bar Harbor, ME, USA) and fed in standard conditions of 12 h of light/day at a room temperature of 22 °C. Twenty male APP/PS1 transgenic mice were randomly divided into two groups, and ten nontransgenic mice were used as a negative control group. THA was dissolved in 0.9% sodium chloride injection containing 5% Tween 80. The two 6-month APP/PS1 transgenic groups were given 300 mg THA/kg per day or vehicle by intragastric administration, and the 6-month non-transgenic group was given vehicle through intragastric administration. After 100 d of administration, the learning ability of the mice was detected using Morris water maze (MWM) assay. Upon completion of the MWM test, all mice were immediately euthanized, and all brains were removed and bisected in the mid-sagittal plane. The left sides of the brains were fixed in 4% paraformaldehyde for histologic analysis, and the right sides of the brains were stored at -80 °C.
MWM assay MWM assay was used to determine the cognitive ability of mice, as described in a previously published report [40] . Briefly, each mouse was given 90 s for 3 trials per day for 9 consecutive days to find the invisible submerged white platform in a circular pool (120 cm in diameter, 50 cm deep) filled with milktinted water using a variety of visual cues located on the pool wall. The mice were allowed to stay on the platform for 15 s if the mice found the platform within 90 s. However, if the mice failed to find the platform within 90 s, the mice were guided to find the platform and were allowed to stay on the platform for 15 s. All data were collected for the mouse performance analysis. The escape latency, which is the amount of time the mouse takes to find the platform, is a major parameter for cognitive evaluation and thus was calculated every day.

Congo red and Thioflavine S staining
The paraformaldehyde-fixed brain tissues (~3.5 mm thick) containing the hippocampus and cortex were embedded in paraffin. Ten-micrometer-thick coronal sections were used for Congo red and Thioflavine S staining, according to a previously described approach [40] .
Nine sections (10 mm thick) were produced per mouse, and each group comprised 4 mice. For Congo red staining, the sections were deparaffinized, hydrated and stained in Congo red for 20 min, and then differentiated in alkaline alcohol solution, followed by counterstaining with Gill's hematoxylin for 30 s, and finally mounting with neutral gum. The total number of Congo red stained plaques in the hippocampus and cortex (4 mice per group) was separately calculated for the statistical Table 1. Primer sequences.

analysis.
For Thioflavine S staining, the sections were deparaffinized, hydrated and stained in 1% Thioflavine S for 5 min, then differentiated in 70% alcohol solution for 5 min, followed by mounting with glycerol gel. The number of Thioflavine S plaques was counted for every three to six fields throughout the entire hippocampus and cortex (4 mice per group) using Image-Pro Plus (Media Cybernetics).

Immunofluorescence staining
The sections described as before [40] , embedded in paraffin, were prepared for immunofluorescence staining. Nine sections (10 mm thick) were produced per mouse, and each group comprised 4 mice. After being deparaffinized and hydrated, the sections were incubated in PBS for 2 min, followed by incubation with 3% hydrogen peroxide (H 2 O 2 ). Then, citric acid (pH 6.0) was used for antigen retrieval of the sections submitted to microwave heating at medium heat for 6 min, followed by natural cooling to room temperature, for 4 times. Then, the sections were blocked with PBS-0.05% Triton-X 100-10% horse serum for 10-30 min and incubated with primary antibody overnight at 4 °C in PBS-0.05% Triton X-100-1% horse serum, and then the sections were incubated in primary antibody at RT for 45 min. The sections were subsequently rinsed with PBS 3 times (3 min/time) and incubated for 45 min in secondary rabbit antibody Alexa Flour ® 488 goat anti-rabbit IgG (H+L) (PBS-0.05%Triton X-100-1% horse serum). The images were acquired using a laser scanning confocal microscope (Nikon, Japan).
Wild-type Drosophila melanogaster culture and lifespan assays yw D melanogaster isogenic Canton-S flies (outcrossed with wlll8 (isoCJ1) for 5 generations) were used in the experiments by JoeKai company. All flies were bred on standard cornmeal food and were originally reared at 23-25 °C and 40%-60% relative humidity (RH) on a 12 h-12 h light-dark cycle. On the second day after eclosion (DAE), male flies were selected and stored separately into different vials (15 flies/vial) for the drug feeding process (25 °C, 40%±15%). Initially, there were at least 225 flies for each condition. One day before drug feeding, approximately 1 mg compound was diluted to 0.05 or 0.005 mg/mL with 4% sucrose and 5% Tween-80 working solutions. At DAE3, the flies were treated with drugs for 4 h after 2 h of starvation, followed by 18 h of rest in a fresh food vial. On each feeding day, 40 μL drug or vehicle solutions were delivered to each vial. Dead flies were recorded on a daily basis until all flies had died. All statistical analysis was performed using SPSS version 20 (IBM).

Statistical analysis
Significant differences between multiple treatments and controls were analyzed using one-way ANOVA followed by Bonferroni's post hoc test. Differences between double variants were analyzed with two-way ANOVA followed by Bonferroni's post hoc test. P values of less than 0.05 were considered to be significant.

THA inhibits both Aβ level and Tau hyperphosphorylation Compound screening
To screen for the natural product extracts that exhibit multifunctional activities in inhibiting both Aβ levels and Tau hyperphosphorylation, an ELISA assay [43] was first applied to screen for extracts active in inhibiting Aβ levels. The ELISA assay was performed using our laboratory's in-house natural product extract library (~230 extracts) in CHO-APP/BACE1 cells, and THA was finally discovered. THA was then determined to exhibit ability to inhibit Tau hyperphosphorylation in both SH-SY5Y and primary neuronal cells.

THA decreased Aβ levels
To evaluate the inhibition of THA against Aβ levels, CHO-APP/BACE1 cells were incubated with different concentrations of THA (20, 10, 1, or 0 μg/mL), and Aβ 40 levels in the cell culture medium were detected by ELISA assay. As shown in Figure 1B, THA treatment markedly decreased Aβ levels in the cells.
THA inhibits Aβ production involving alleviation of PERK/eIF2α signaling-mediated BACE1 expression THA inhibited Aβ production Given that the Aβ level involves a balance of Aβ production and clearance [44] , we investigated whether the THA-induced Aβ inhibition was achieved through inhibition of Aβ production and/or promotion of Aβ clearance. We first examined the level of sAPPβ, a direct product of APP upon β-cleavage [44] , in CHO-APP/BACE1 cells by ELISA assays. As indicated in Figure 2A, THA effectively reduced sAPPβ in the CHO-APP/ BACE1 cells, indicative of its inhibition of Aβ production.

THA inhibited Aβ production involving alleviation of PERK/eIF2α signaling-mediated BACE1 expression
Next, we sought to explore the mechanism underlying the THA-induced suppression of Aβ production. Given that BACE1 is the key rate-limiting enzyme responsible for Aβ production [4] , we focused on the regulation of THA against BACE1 relating to its enzymatic activity and protein expression. Interestingly, THA had no effects on BACE1 enzymatic activity in vitro ( Figure 2B, TDC as a positive control [40] ) but decreased sAPPβ and BACE1 protein levels in CHO-APP/ BACE1 cells (Figure 2C, 2F) and primary astrocytes ( Figure  2D, 2G), as indicated by Western blot results. Accordingly, we investigated the THA-mediated regulation of BACE1 transcription and/or translation in response to its inhibition against BACE1 expression. Considering that THA had no effects on BACE1 transcription, according to the RT-PCR assay ( Figure 2E), we focused on the regulation of THA against BACE1 translation. In light of the fact that PERK/ eIF2α signaling is highly implicated in BACE1 translation [45] , we inspected whether THA regulated PERK/eIF2α signaling. As expected, the Western blot results demonstrated that THA diminished the phosphorylation of PERK and eIF2α in CHO-APP/BACE1 cells ( Figure 2C, 2F) and astrocytes ( Figure 2D,  2G).
Thus, our results indicated that THA inhibited Aβ production and this process involved the alleviation of PERK/eIF2α signaling-mediated BACE1 expression.
THA promotes Aβ clearance involving activation of both PI 3 K/ AKT/mTOR and AMPK/Raptor/mTOR signaling-mediated autophagy THA promoted Aβ clearance Next, we investigated the potential of THA in the promotion of Aβ clearance. The assay was carried out in SH-SY5Y cells ( Figure 3A, 3B), BV 2 cells (Figure 3C, 3D) and primary astrocytes ( Figure 3E, 3F). As indicated, THA markedly enhanced exogenous Aβ 42 /Aβ 40 clearance in the cells, indicative of the promotion of THA on Aβ clearance. According to a published report, Cu 2+ binds to Aβ [46,47] and promotes fiber formation [48] . Here, to exclude the possibility that the inhibition of exogenous Aβ by THA treatment was a result of a direct reaction between Aβ and THA, we performed the related ELISA assays by using Cu 2+ as a positive control in vitro. As demonstrated in Supplementary Figure S1A, incubation of THA did not reduce exogenous Aβ, and Cu 2+ treatment markedly reduced Aβ levels. This result thereby implied that the THA-induced inhibition of exogenous Aβ was not due to a direct extracellular reaction between Aβ and THA.
In addition, to prove that THA did not impair the cellular function of Aβ endocytosis resulting in the decline of "intracel- lular" Aβ, we also performed the relevant assays in SH-SY5Y cells, in which we examined Aβ levels in the supernatant and cell lysis by ELISA (Supplementary Figure S1B, S1C, Figure  3A, 3B). The results indicated that THA reduced Aβ levels both in the supernatant (Supplementary Figure S1B, S1C) and the cell lysis ( Figure 3A, 3B), which thus indirectly demonstrated that THA rendered no impairment on the cellular function of Aβ endocytosis.

THA activated autophagy
Given that autophagy, a major degradation process for abnormal and aggregated proteins, plays an important role in Aβ clearance [49] , we next investigated the potential of THA in the regulation of autophagy.
In addition, Western blot assay was also performed to further verify the activation of THA on autophagy in SH-SY5Y cells ( Figure 4C, 4F), BV 2 cells (Figure 4D, 4G) and primary astrocytes ( Figure 4E, 4H) by testing several key marker proteins of autophagy, including Unc51-like kinase 1 (ULK1), LC3II and polyubiquitin binding protein p62 [52] . The results demonstrated that THA reduced phosphorylated ULK1 and increased LC3II protein levels in all three tested cells. It was noted that THA suppressed p62 levels in both SH-SY5Y cells and primary astrocytes, but had no effects on BV 2 cells, possibly due to the cellular selectivity of THA, similar to the case of a previously published report [43] . Thus, these results indicated that THA activated autophagy in the cells.

THA alleviates Aβ levels involving autophagy activation
Next, ELISA assay was carried out to verify the activity of THA in reducing Aβ levels via autophagy activation by incubating cells with the autophagy inhibitor chloroquine (CQ [53] ). In the assay, CHO-APP/BACE1 cells were treated with THA (20 μg/mL), vehicle alone, CQ (10 μmol/L), or THA in combination with CQ for 24 h. The results in Figure 4I demonstrated that CQ effectively reversed the THA-induced decrease in Aβ levels. This result indicated that THA reduced Aβ levels involving autophagy activation.

THA-induced autophagy promotion involves both PI 3 K/AKT/mTOR and AMPK/Raptor/mTOR signaling pathways
Because we determined the capability of THA in stimulating autophagy, we next explored its underlying mechanisms. Given the report that the PI 3 K/AKT/mTOR and AMPK/ Raptor/mTOR signaling pathways are tightly implicated in autophagy [54] , we carried out the related assays to investigate the potential regulation of THA against these two pathways in response to the THA-induced autophagy stimulation in SH-   As expected, the results demonstrated that THA decreased the phosphorylation of mTOR, PI 3 K and AKT, whereas it increased the phosphorylation of AMPK and Raptor in all three tested cells. THA suppressed P70S6K phosphorylation in both SH-SY5Y cells and primary astrocytes but had no effects on BV 2 cells, probably due to the cellular selectivity of THA. Thus, these results indicated that THA activated autophagy by regulating both the PI 3 K/AKT/mTOR and AMPK/Raptor/mTOR signaling pathways.

THA functions as both a PI 3 K inhibitor and an indirect AMPK activator
Because we determined that THA inhibited PI 3 K phosphorylation and stimulated AMPK phosphorylation, we next investigated whether THA functioned as an inhibitor against the PI 3 K enzyme and as an activator of the AMPK enzyme. In vitro enzymatic activity assays were carried out, which demonstrated that THA directly inhibited PI 3 K enzyme activity but rendered no effects on AMPK enzyme ( Figure 5N, 5O. Wortmannin: PI 3 K inhibitor [55] ; A769662: AMPK activator [56] ). Therefore, THA functioned as both a PI 3 K inhibitor and an indirect AMPK activator.

THA reduces Tau hyperphosphorylation by functioning as a GSK3β inhibitor
It was reported that glycogen synthase kinase 3β (GSK3β) and cyclin-dependent kinase 5 (CDK5) are the two main kinases for Tau pathogenesis [14,15] . GSK3β is activated when its Tyr216 is phosphorylated or its Ser9 is de-phosphorylated, thus enhancing Tau phosphorylation [14] . CDK5 is activated by p35 or p25 (the cleaved product of p35) to stimulate Tau phosphorylation [15]. Given these facts, we next performed Western blot assays to investigate the regulation of THA against GSK3β and CDK5. The results indicated that THA effectively decreased GSK3β Tyr216 phosphorylation without affecting its Ser9 phosphorylation and failed to affect CDK5 or p35/p25 in SH-SY5Y cells ( Figure 6A, 6D), BV 2 cells ( Figure 6B, 6E) or primary astrocytes ( Figure 6C, 6F).
Furthermore, in vitro CDK5 and GSK3β enzyme activity assays were also performed. The results demonstrated that HTA had no effects on CDK5 enzymatic activity ( Figure 6G, PHA-793887: CDK5 inhibitor [41] ) but inhibited GSK3β enzymatic activity with an IC 50 of 1.32±0.85 μg/mL ( Figure 6H). Therefore, all these results suggested that THA reduced Tau phosphorylation by functioning as a GSK3β inhibitor.
THA ameliorates space learning ability in APP/PS1 transgenic mice It has been reported that APP/PS1 transgenic mice may express chimeric mouse/human Swedish mutant amyloid precursor protein and mutant human presenilin 1 protein and that they exhibit obvious age-related memory impairments and Aβ accumulation and deposition. These mice have been largely regarded as an appropriate model for AD-related research [57] . Given that THA was effective in both decreasing Aβ content and attenuating Tau pathology in cells, we next examined the potential of THA in the improvement of space learning ability in APP/PS1 transgenic mice by performing a MWM assay [58] .
The results demonstrated that in the training trials, the escape latencies of the non-transgenic vehicle group to find the platform were significantly shorter compared with the transgenic AD model vehicle group (Figure 7A, 7B), indicating that the space learning ability of the transgenic AD model mice were obviously impaired. However, the latencies of the THAadministered (300 mg·kg -1 ·d -1 ) transgenic AD model group were significantly decreased compared with the transgenic vehicle group (Figure 7A, 7B). We chose two doses of THA, 100 and 300 mg·kg -1 ·d -1 , for this study. Because THA at 100 mg·kg -1 ·d -1 exhibited no effect, we omitted this dosage in the text. These results suggested that THA ameliorated the space learning ability of APP/PS1 transgenic mice.
THA reduces senile plaque formation in APP/PS1 transgenic mice Congo red and Thioflavine S staining assays Aβ-formed senile plaques are one of the major hallmarks of AD, and the progressive deposition of these plaques in the hippocampus and cortex results in space learning memory impairment [59] . As our results demonstrated that THA ameliorated space learning impairments in APP/PS1 transgenic mice, we next inspected whether THA alleviated senile plaque formation in the hippocampus and cortex of the tested transgenic mice by Congo red (Figure 8A-8D) and Thioflavine S staining ( Figure 8E-8H) assays.
After the MWM test, the mice were sacrificed, and the left sides of the brains were immediately fixed in 4% paraformaldehyde for histological analysis. As expected, the numbers of Congo red ( Figure 8A-8D)-stained senile plaques in both the hippocampus and cortex of the vehicle-administered transgenic mice were greater than in the vehicle-administered non-transgenic mice. However, treatment with THA (300 mg·kg -1 ·d -1 ) could effectively reverse the high senile plaque numbers in both the hippocampus and cortex of the transgenic mice. Similarly, the Thioflavine S staining assay results (Figure 8E-8H) also confirmed the inhibition of THA against senile plaque formation in the transgenic mice. The senile plaque burdens stained by Thioflavine S in the hippocampus and cortex of the vehicle-administered transgenic mice were markedly more severe compared with those of vehicle-administered non-transgenic mice, whereas treatment with THA reversed the senile plaque formation in the transgenic mice.

ELISA assay
To further investigate the inhibitory activity of THA on Aβ-formed senile plaques, we also inspected the effect of THA on Aβ 40 /Aβ 42 levels in hippocampal and cerebral cortical homogenates by an ELISA assay. As shown in Figure 8I-8L, Aβ 40 /Aβ 42 levels in both the hippocampus and cerebral cortex of the transgenic mice were higher compared with those  Figures (J-L). (N) An AMPK enzyme reaction system was directly incubated with different concentrations of THA (20 or 2 μg/mL), and the in vitro AMPK activity was then evaluated. A769662: AMPK activator [56] . (O) A PI 3 K enzyme reaction system was directly incubated with different concentrations of THA (40, 20, 10, or 0 μg/mL), and the in vitro PI 3 K activity was then evaluated. Wortmannin: PI 3 K inhibitor [55] ; GAPDH was used as a loading control in the Western blot assays. The results were obtained from three independent experiments. Values are the mean±SEM, one-way ANOVA, Bonferroni's multiple comparison test. n=3. * P<0.05, ** P<0.01 compared with the control group, control group: 0 μg/mL THA treatment group. Li C et al Acta Pharmacologica Sinica  [41] . (H) GSK3β enzyme reaction system was directly incubated with different concentrations of THA (40, 20, 10, 1, 0.1, 0.001, or 0.0001 μg/mL), and the in vitro GSK3β activity was then evaluated. THA inhibited GSK3β enzymatic activity with an IC 50 of 1.32±0.85 mg/L. GAPDH was used as a loading control in the Western blot assays. The results correspond to three independent experiments. Values are the mean±SEM, one-way ANOVA, Bonferroni's multiple comparison test. n=3. ** P<0.01 compared with the control group, control group: 0 mg/L THA treatment group.
of the non-transgenic mice, and administration of THA (300 mg·kg -1 ·d -1 ) efficiently decreased Aβ 40 /Aβ 42 levels in both the hippocampus and cerebral cortex of the transgenic mice compared with those of the vehicle-treated transgenic group. In addition, we also performed Western blot assay [60,61] to detect the effect of THA on Aβ polymer in the cells and the cortex (Supplementary Figure S1D-S1G), and the results indicated that THA had no effects on Aβ polymer. Thus, these results suggested that THA may downregulate soluble Aβ to reduce the levels of Aβ plaques in APP/PS1 transgenic mice.
THA alleviates PERK/eIF2α-mediated BACE1 translation in response to Aβ production inhibition in APP/PS1 transgenic mice Given that THA repressed PERK/eIF2α-mediated BACE1 translation in response to Aβ production inhibition in cells, we next confirmed the effect in APP/PS1 transgenic mice.
In the assay, we detected the effects of THA on the protein level of sAPPβ/BACE1 and on the phosphorylation of PERK/ eIF2α in the cortices of the tested mice by Western blotting. Figure 9A, 9B, the protein level of sAPPβ/ BACE1 and the phosphorylation of PERK/eIF2α in the transgenic mice were higher than those in the non-transgenic mice, whereas treatment with THA apparently inhibited those effects compared with the THA-non-treated transgenic group. Next, we carried out an RT-PCR assay to investigate if THA inhibits BACE1 transcription and found that THA failed to affect BACE1 mRNA levels ( Figure 9C). These results indicated that THA alleviated PERK/eIF2α-mediated BACE1 translation in response to Aβ production inhibition in APP/ PS1 transgenic mice, in line with the corresponding cell-based results.

As indicated in
THA stimulates both PI 3 K/AKT/mTOR-and AMPK/Raptor/mTORmediated autophagy in APP/PS1 transgenic mice Because THA has been determined to stimulate PI 3 K/AKT/ mTOR-and AMPK/Raptor/mTOR-mediated autophagy in the promotion of Aβ clearance in cells, we next inspected such effects in APP/PS1 transgenic mice. In the assay, we focused on the cortex of the tested mice by Western blotting.

THA stimulated autophagy
To examine the stimulation of THA against autophagy in vivo, we at first investigated the regulation of THA against the key proteins involved in the autophagy pathway. The results shown in Figure 9D, 9E revealed higher protein levels of p62 and phosphorylated ULK1 and lower protein levels of LC3II in vehicle-treated transgenic mice compared with those in vehicle-treated non-transgenic mice, which indicated the impaired autophagy process in transgenic mice [62] . Notably, THA administration obviously reversed all of these effects, thereby implying that THA could activate autophagy in APP/ PS1 transgenic mice.

THA stimulated PI 3 K/AKT/mTOR-mediated autophagy
Next, we examined the enhancement of THA on PI 3 K/AKT/ mTOR-mediated autophagy in vivo. As expected, Western blot results ( Figure 9F-9I) indicated higher phosphorylation levels of PI 3 K, AKT, mTOR and P70S6K in vehicle-treated transgenic mice compared with the levels in vehicle-treated nontransgenic mice. Indicative of the activated PI 3 K/AKT/mTOR pathway in response to the impaired autophagy in APP/PS1 transgenic mice [41] , administration of THA could obviously reverse all of the effects in the transgenic mice. These results thereby suggested that THA stimulated PI 3 K/AKT/mTORmediated autophagy.

THA stimulated AMPK/Raptor/mTOR-mediated autophagy
Similarly, the Western blot results ( Figure 9J, 9K) also indicated that administration of THA could activate the phosphorylation of AMPK/Raptor in APP/PS1 transgenic mice. Interestingly, the phosphorylation levels of AMPK and Raptor in the vehicle-treated transgenic mice were at similar levels as in non-transgenic mice.
Taken together, these results indicated that THA stimulated PI 3 K/AKT/mTOR-and AMPK/Raptor/mTOR-mediated THA reduces Tau hyperphosphorylation involving inhibition of GSK3β in APP/PS1 transgenic mice Given the published reports on the Tau-relevant study in APP/PS1 transgenic mice [34,63] , we also performed a Taurelated assay in the transgenic mice. In the assay, we investigated the potential of THA to inhibit Tau hyperphosphorylation in vivo by performing an immunohistochemical assay. As shown in Figure 10A-10D, Tau hyperphosphorylation bur-  dens in the hippocampus and cortex of vehicle-administered transgenic mice were more severe than in the vehicle-administered non-transgenic mice group, and THA administration effectively reversed these effects in the transgenic mice. Subsequently, a Western blot assay was performed. Similar to the cell-based assays, we investigated the effects of THA on GSK3β phosphorylation at Ser9/Tyr216 and CDK5 and p35/ p25 protein levels in the transgenic mice. As shown in Figure  10E, 10F, an approximately 40% change in p-Tau/Tau between wild-type and AD mice was determined, and this result was close to a published report where a 50% change in p-Tau/Tau between wild-type and AD mice was found [64,65] . In addition, THA administration stimulated the inactive form of GSK3β (phosphorylated at Ser9) and inhibited the active form of GSK3β (phosphorylated at Tyr216) in APP/PS1 transgenic mice, indicating that THA could inhibit GSK3β activity in vivo. As expected, THA had no effects on CDK5 or p35/p25 in the mice ( Figure 10E, 10F). Thus, the results demonstrated that THA reduced Tau phosphorylation by inhibition of GSK3β in the APP/PS1 transgenic mice, in consistence with the cellbased results.
THA prolongs the lifespan of wild-type Drosophila melanogaster Accumulating evidence has revealed that aging is highly associated with AD, and delaying aging is an applicable strategy for decreasing AD rates and postponing AD progression [66] , while autophagy is tightly linked to lifespan [67] . Given these facts, we thus investigated the potential of THA to extend the lifespan of Drosophila melanogaster. As expected, the results indicated that THA treatment (0.05 mg/mL) effectively extended the lifespan of Drosophila melanogaster ( Figure 11). This result thereby supported that THA may prevent AD by delaying aging.

Discussion
Along with the fast pace of an aging society are aging diseases (eg, AD, type 2 diabetes, cancers, etc), which severely afflict the aged population. In fact, the neurodegenerative disease AD has been clinically accepted as one of the leading causes of death [24] . It is characterized by progressive memory loss and cognitive and behavioral disturbances, and there is no efficient treatment against this disease, although there have been clinical drugs capable of improving the symptoms of AD [68] . Considering the complicated pathogenesis of AD, multi-targets have become a focus in the discovery of drug compounds for treatment of this disease [18] . In the current work, given the diversities in both the chemical structures and acting targets for natural products, we established a multi-target strategy for discovering active reagents capable of suppressing both Aβ levels and Tau hyperphosphorylation from natural products. We found that the extract of Thamnolia vermicularis improves learning ability in APP/PS1 transgenic mice by ameliorating both Aβ and Tau pathologies, which highly supports the efficiency of our strategy for the discovery of potential anti-AD agents.
Recently, several pharmacological functions have been discovered for the lichen Thamnolia vermicularis in the treatment of various diseases, including hypertension, cough, and neurasthenia, and for its antioxidant and anticancer properties [20,22] . Therefore, our current results concerning its anti-AD activity have also expanded the new pharmacological functions for this natural product. Various types of pure small molecules have been separated from Thamnolia vermicularis [69] , but no report has yet been published regarding their potential multitarget function in the treatment of AD. Considering the relatively high contents of squamatic acid (42%) and baeomycesic acid (46%) ( Figure 1A) in THA, we also examined the potential activity of these two compounds in the regulation of Aβ accumulation and/or Tau phosphorylation, and the results indicated that neither of these two compounds exhibited effects (data not shown). Currently, few reports have been published on the functions of baeomycesic acid and squamatic acid [70,71] , and baeomycesic acid was determined to exhibit antiproliferative effects on several human tumor cell lines [72] . Thus, it is expected that the current study may also provide chemical direction for the discovery of compounds with multi-target activities in the repression of Aβ/Tau pathology from Thamnolia vermicularis.
In summary, we reported that the ethanol extract of Thamnolia vermicularis (THA) improved learning ability in APP/PS1 transgenic mice by inhibiting both Aβ levels and Tau hyperphosphorylation. In addition, THA as an effective autophagy activator, also efficiently prolonged the lifespan of wild-type Drosophila melanogaster, thus exhibiting potential in the prevention of AD by prolonging aging. Figure 12 shows a proposed model for the amelioration of Aβ/Tau pathology by THA. THΑ stimulated either PI 3 K/AKT/mTOR-or AMPK/raptor/ mTOR signaling-mediated autophagy in promotion of Aβ clearance as both a PI 3 K inhibitor and an AMPK indirect activator and restrained Aβ production as a suppressor against PERK/eIF2α-mediated BACE1 expression. Additionally, as a GSK3β inhibitor, THA inhibited Tau hyperphosphorylation. Therefore, our results have highlighted the potential of THA in the treatment of AD.

Author contribution
Xu SHEN, Zhi-yuan ZHU, and Cong LI designed the study; Cong LI carried out the experiments and analyzed the data; Min LEI and Li-hong HU synthesized the compound; Zhiyuan ZHU, Xiao-fan SHI, and Xiao-dan GUO participated in animal experiments; Xu SHEN and Cong LI wrote the manuscript; all authors, including Tie-qiao WEN, Jia-yi WU, Jiazhen JIN, and Vatcharin RUKACHAISIRIKUL, discussed the results and contributed to the revision of the final manuscript.

Supplementary information
Supplementary figures are available on the website of Acta Pharmacologica Sinica.  Tau are believed to be two major hallmarks of AD pathology. THΑ stimulated either PI 3 K/AKT/mTOR-or AMPK/raptor/mTOR signaling-mediated autophagy in promoting Aβ clearance as both a PI 3 K inhibitor and an AMPK indirect activator. THA restricted Aβ production as a suppressor against PERK/eIF2α-mediated BACE1 expression. Additionally, THA functioned as a GSK3β inhibitor, repressing Tau hyperphosphorylation.