Gypenoside XVII Enhances Lysosome Biogenesis and Autophagy Flux and Accelerates Autophagic Clearance of Amyloid-β through TFEB Activation

Abstract

A strategy for activating transcription factor EB (TFEB) to restore autophagy flux may provide neuroprotection against Alzheimer’s disease. Our previous study reported that gypenoside XVII (GP-17), which is a major saponin abundant in ginseng and Panax notoginseng, ameliorated amyloid-β (Aβ)_25-35-induced apoptosis in PC12 cells by regulating autophagy. In the present study, we aimed to determine whether GP-17 has neuroprotective effects on PC12 cells expressing the Swedish mutant of APP695 (APP695swe) and APP/PS1 mice. We also investigated the underlying mechanism. We found that GP-17 could significantly increase Atg5 expression and the conversion of LC3-I to LC3-II in APP695 cells, which was associated with a reduction in p62 expression. GP-17 also elevated the number of LC3 puncta in APP695 cells transduced with pCMV-GFP-LC3. GP-17 promoted the autophagy-based elimination of AβPP, Aβ₄₀, and Aβ₄₂ in APP695swe cells and prevented the formation of Aβ plaques in the hippocampus and cortex of APP/PS1 mice. Furthermore, spatial learning and memory deficits were cured. Atg5 knockdown could abrogate the GP-17-mediated removal of AβPP, Aβ₄₀, and Aβ₄₂ in APP695swe cells. GP-17 upregulated LAMP-1, increased LysoTracker staining, and augmented LAMP-1/LC3-II co-localization. GP-17 could release TFEB from TFEB/14-3-3 complexes, which led to TFEB nuclear translocation and the induction of autophagy and lysosome biogenesis and resulted in the amelioration of autophagy flux. The knockdown of TFEB could abolish these effects of GP-17. In summary, these results demonstrated that GP-17 conferred protective effects to the cellular and rodent models of Alzheimer’s disease by activating TFEB.

Keywords

APP/PS1 mice autophagy flux lysosome biogenesis transcription factor EB

INTRODUCTION

Alzheimer’s disease (AD) is the most common neurodegenerative disorder in the elderly population characterized by the progressive loss of memory and the generation of extracellular senile plaques composed mainly of aggregated amyloid-β (Aβ), neurofibrillary tangle, and neuronal degeneration in the brain [1]. According to the amyloid hypothesis, Aβ is derived from the sequential cleavage of amyloid-β protein precursor (AβPP) by β-site AβPP-cleaving enzyme 1 and the γ-secretase complex. An imbalance between the formation and removal of Aβ resulted in toxic Aβ aggregates, which are strongly associated with neurodegeneration and cognitive impairment in AD [2]. Therefore, the efficient removal of excessive AβPP and Aβ is important for the prevention and treatment of AD.

Macroautophagy (referred to as autophagy hereafter) is a highly evolutionarily conserved cellular self-digestion process with essential functions in the degradation of AβPP and Aβ [3]. Autophagy is a degradation pathway whereby cytosolic double membrane-bound compartments called autophagosomes engulf and sequester cytoplasmic constituents. These autophagosomes then fuse with lysosomes and forms autolysosomes. This phenomenon is termed as autophagy flux, which results in the degradation of the engulfed components. However, several lines of evidence raise the possibility that fundamental defects occur in autophagy flux because lysosomal dysfunction is an important step in AD development [4, 5]. In fact, maintaining autophagy flux by restoring lysosome biogenesis rather than increasing autophagosome formation alone have resulted in the efficient degradation of Aβ and provided therapeutic effects to cellular and rodent models of AD [6, 7].

Transcription factor EB (TFEB) is the only known transcription factor that is a master regulator of lysosome biogenesis [8]. Under normal conditions, TFEB proteins are usually sequestered in the cytosol in a transcriptionally inactive form by interaction with 14-3-3 proteins, thus preventing the cytoplasm-to-nucleus shuttling of TFEB [9]. Conversely, during nutrient starvation [10], inhibition of the mammalian target of rapamycin complex 1 (mTORC1) [11], or pharmacological intervention [12], the interaction between TFEB and 14-3-3 proteins was diminished, thus resulting in the nuclear accumulation of TFEB. Activated TFEB binds to target genes that bear consensus-coordinated lysosomal expression and regulation elements, thus resulting in the transcriptional induction of target genes including lysosomal-associated membrane protein 1 (LAMP-1) and phosphatase and tensin homolog (PTEN) [13, 14]. In this respect, TFEB is at the crossroads of the regulatory mechanisms that coordinate autophagy with lysosome biogenesis. Insights from rodent models indicate that overexpressing TFEB or inducing the nuclear translocation of TFEB could promote autophagic clearance of Aβ. As a result, TFEB activators may offer more superior benefits than autophagy inducers. TFEB might be an attractive novel therapeutic target for AD.

There is a growing body of evidence to support the fact that phytoestrogens such as genistein and daidzein could activate TFEB [12, 15]. We have previously reported that GP-17, a novel phytoestrogen isolated from ginseng, P. notoginseng, and G. pentaphyllum, had neuroprotective effects against Aβ_25–35-induced apoptosis in PC12 cells by modulating autophagy [16]. However, our previous study monitored only the changes in intracellular microtubule-associated protein 1 light chain 3 (LC3) puncta and the expression of LC3-II. It is still unclear whether GP-17 accelerates autophagic clearance of Aβ through TFEB activation in cellular and rodent models of AD.

Considering the above evidence, we focused on the involvement of TFEB in the neuroprotective effects of GP-17 against PC12 cells expressing the Swedish mutant of APP695 (APP695swe) and APP/PS1 mice, which are the two well-known cellular and rodent models of AD. We found that GP-17 accelerated the autophagy-based elimination of Aβ in vivo and in vitro through TFEB activation.

MATERIALS AND METHODS

Cell culture

PC12 cells were obtained from the Cell Resource Center of the Institute of Basic Medical Sciences, Peking Union Medical College and Chinese Academy of Medical Sciences (Beijing, China). PC12 cells were cultured in DMEM supplemented with 10% horse serum and 5% FBS at 37°C in 5% CO₂ and 95% atmosphere. To establish APP695swe cells, the full-length cDNA fragment of APP695swe was amplified by PCR and was cloned into pcDNA3.1 (+) vector to construct the recombinant plasmid pcDNA3.1-APP695swe. PC12 cells were transduced with pcDNA3.1-APP695swe or the control vectors using Lipofectamine 2000, and stable transfectants were selected in 0.8 mg/mL G418. The expression of gene and protein was examined by RT-PCR and immunoblotting, respectively. APP695swe cells were maintained in DMEM supplemented with 10% horse serum, 5% FBS, and G418 (0.2 mg/mL final concentration) at 37°C in a 5% CO₂ incubator. In all experiments, the APP695swe cells in the exponential phase of passages were used. For the autophagic analysis, when 60% confluence was reached, the cells were transiently transfected for 48 h with Atg5 siRNA, TFEB siRNA, or pCMV-GFP-LC3, and with equivalent concentrations of control siRNA or control vectors using Lipofectamine 2000. GP-17 stock solution (1 M) was prepared in DMSO and was diluted with fresh complete medium immediately before use. Unless otherwise indicated, control and APP695swe cells were incubated with 10 μM GP-17 or DMSO (final concentration was < 0.1%) for 12 h.

Transmission electron analysis

Cells were fixed in 4% paraformaldehyde and 1% glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.2) overnight at room temperature. Following fixation, cells were treated with reduced 1% osmium tetroxide, followed by 1% tannic acid in 0.1 M sodium cacodylate buffer for 1 h. The cells were then stained with 2% aqueous solution of uranyl acetate for 30 min, dehydrated in a series of graded ethanol concentrations, and processed for enface embedding in PolyBed (Polysciences). Blocks were sectioned at a 90 nm thickness, poststained with Venable’s lead citrate, and viewed with a transmission electron microscope (JEOL, Tokyo, Japan). Images were obtained on a digital camera (Advanced Microscopy Techniques, MA, USA) by observers who were blinded to the experimental groups.

LysoTracker staining

Lysosome abundances were assessed by LysoTracker Red DND-99 staining. Cells were incubated with LysoTracker Red DND-99 (50 nM final concentration) at 37°C for 30 min. Following fixation in 4% paraformaldehyde, the cells were counterstained with DAPI. The LysoTracker signal was excited at 562 nm, and emission was read at 595 nm using a laser scanning confocal immunofluorescence microscope (Leica, Germany).

Flow cytometry analysis

Control and APP695swe cells were co-treated with GP-17 (10 μM) and 3-methyladenine (3-MA, 2 mM) or bafilomycin A1 (50 nM) for 12 h. The endogenous LC3 was stained using rat anti-LC3B (#2775, Cell Signaling Technology; 1 : 100), washed with PBS containing 0.05% saponin, and processed for flow cytometry analysis on a FACScan instrument (Becton-Dickinson, CA, USA) for cells containing a saponin resistant LC3-II.

Co-IP

The interaction between TFEB and 14-3-3 was detected by Co-IP Kit. Briefly, cells were lysed in ice-cold IP lysis buffer supplemented with protease and phosphatase inhibitors cocktail for 10 min. Cell lysates were centrifuged at 12,000 rpm for 10 min at 4°C, and the supernatant was collected. The supernatant was pre-cleared using the control agarose resin. An amount of 1 mg protein was incubated with 10 μg antibody immobilization AminoLink Plus coupling resin in 10 μL of 20X coupling buffer, 180 μL of ultrapure water, and 3 μL of the sodium cyanoborohydride solution on a rotator overnight at 4°C. Immunoprecipitates were washed four times with lysis buffer, and proteins were eluted with elution buffer. The sample was heated at 95– 100°C for 5 min with lane marker sample buffer and was analyzed by immunoblotting.

Animals and treatment

All animal procedures were conducted in accordance with the National Institutes of Health Guidelines on the Use and Care of Animals, with approval from the Institutional Animal Experiment Committee of Peking Union Medical University. All efforts have been made to minimize animal suffering and the number of animals used. Five-month-old male double-transgenic APPswe/PS1dE9 (APP/PS1) and non-transgenic (wild type, WT) littermates were purchased from the Institute of Laboratory Animal Science, Chinese Academy of Medical Science (Beijing, China). They were housed in animal care facilities under controlled conditions of temperature (23 ± 1°C), humidity (50 ± 10%), with an alternating 12 h light/dark cycle and free access to food and water. After 7 d of environmental adaption, APP/PS1 mice (n = 40) and WT littermates (n = 40) were randomly assigned into vehicle (sterile distilledwater) -treated WT (n = 20), GP-17 (40 mg/kg/day, by gavage for 60 d) -treated WT (n = 20), vehicle-treated APP/PS1 mice (n = 20), and GP-17-treated APP/PS1 group (n = 20). The general health of the mice was carefully monitored, and no significant difference was found in body weight as well as the food and water intake between vehicle-treated and GP-17-treated groups.

Morris water maze test

After 60 d of administration, Morris water maze task was conducted to evaluate the spatial learning and memory of mice by technicians who were blinded to the genotypes and experimental groups. Before Morris water maze task began, 30 min of habituation time was given for mice. In maze trials, mice had received 4 training trials with a 15 min interval per day in 5 consecutive d. During each trial, mice (n = 20 per group) were released from 4 semi-randomly assigned starting points and were trained to locate a hidden platform submerged 1 cm below the water surface within 60 s. If a mouse failed to find the platform within 60 s, then the mouse was gently guided to the platform and was allowed to rest on the platform for 15 s. The swimming paths and the escape latency was recorded in each trial. On day 6, the probe trials were performed with the platform removed. During each trial, mice were released from the opposite quadrant and were allowed to swim for 60 s to search for the target platform. The swimming path, the time spent on the target quadrant, and the number of platform crossing were recorded by a computer-controlled video acquisition system (Beijing Sunny Instruments Inc.).

Brain tissue preparation

At the end of treatment, the mice were anesthetized with a mixture of ketamine (100 mg/kg body weight) and xylazine (10 mg/kg body weight). Mice (n = 14 per group) were perfused transcardially with 0.9% saline (pH 7.4), and the brains (n = 6 per group) were removed rapidly and carefully for ELISA; the hippocampal and cortical regions of mouse brains (n = 8 per group) were dissected for western blotting. For immunofluorescence analysis, mice (n = 6 per group) were perfused transcardially with 0.9% saline, followed by ice-cold 4% paraformaldehyde (pH 7.4). Excised brains were immersed in 4% paraformaldehyde overnight and 30% sucrose for 48 h. The brains were embedded in optimum cutting temperaturecompound in a freezing microtome, and 2 μM-thick sections were cut on freezing microtome (Leica,Germany).

ELISA

The levels of Aβ₄₀ and Aβ₄₂ were detected using ELISA kits by technicians who were blinded to the experimental groups. For the detection of Aβ₄₀ and Aβ₄₂ levels in cells, cells were washed, trypsinized, and lysed in RIPA buffer containing phosphatase inhibitor and complete protease inhibitor cocktail. Intracellular Aβ₄₀ and Aβ₄₂ were then detected. The volume of medium used was adjusted to protein concentrations measured in total cell lysates. For the detection of soluble or insoluble Aβ₄₀ and Aβ₄₂ in brain of WT and APP/PS1 mice, dissected tissue was homogenized in RIPA buffer containing phosphatase inhibitor and complete protease inhibitor cocktail. The homogenates were centrifuged at 20,000 rpm for 10 min at 4°C, and the supernatants were pooled for the analysis of soluble Aβ₄₀ and Aβ₄₂. To extract fibrillar and membrane-bound insoluble Aβ₄₀ and Aβ₄₂, the pellets were homogenized in 70% formic acid and were centrifuged at 40,000 rpm for 10 min at 4°C. The supernatants were neutralized with 1 M Tris-base and were analyzed for insoluble Aβ₄₀ and Aβ₄₂.

Immunofluorescence

Cells were fixed in 4% paraformaldehyde for 10 min, permeabilized with 0.1% Triton X-100 for 5 min, and blocked with BSA for 30 min at room temperature. For the detection of TFEB sub-cellular localization, cells were incubated with goat anti-TFEB (ab2636, Abcam; 1 : 100). For the detection of LC3/LAMP-1 co-localization, a mixture of the following antibodies was used: rat anti-LC3B (#2775, Cell Signaling Technology; 1 : 100) and mouse anti-LAMP-1 (ab25630, Abcam; 1 : 100). For the detection of Aβ plaques in hippocampus and cortex, the brain sections were incubated overnight with mouse anti-Aβ (ab11132, Abcam; 1 : 100) after permeabilization and blocking. After washing, the cells and sections were incubated with corresponding secondary antibodies conjugated to Alexa Fluor 488 or Alexa Fluor 647 (Molecular Probes, OR, USA) for 1 h at room temperature. Cells were counterstained with DAPI, and the images were captured and analyzed by a high-content screening system (Molecular Devices, CA, USA). The number of Aβ plaques in the hippocampus and cortex was measured usingImage J.

Western blotting

Western blotting was performed to determine the protein expression in the hippocampus, cortex, or cells according to previously published methods [16] by technicians who were blinded to the experimental groups. Blots were probed with the following antibodies: β-actin (sc-8432) and Lamin B (sc-6216) were obtained from Santa Cruz Biotechnology and were used in 1 : 500 dilutions. Antibodies against LC3B (#2775) and Atg5 (#8540) were obtained from Cell Signaling Technology, and antibodies against AβPP (ab32136), TFEB (ab2636), 14-3-3 (ab9063), PTEN (ab32199), LAMP-1 (ab24170), and Sequestosome 1 (SQSTM1)/p62 (ab56416) were obtained from Abcam and were used in 1 : 1,000dilutions.

Statistical analysis

All data were presented as mean ± standard error of the mean. When data were normally distributed, data were analyzed by unpaired two-tailed Student’s t test for assessing significant differences between two groups, one-way analysis of variance (ANOVA) for multiple groups, and two-way ANOVA for multiple groups with two variables, followed by post hoc Bonferroni’s test for data with equal variances and Dunnett’s T3 for data with unequal variance. When data were not normally distributed, nonparametric tests were used. A two-tailed P value less than 0.05 was deemed statistically significant.

RESULTS

GP-17 facilitated autophagic clearance of Aβ in APP695swe cells

To investigate the effect of GP-17 on Aβ generation, PC12 and APP695swe cells were exposed to 10 μM GP-17 for increasing periods of time. The dose of GP-17 used in the present study was based on our previous study [16]. As shown in Fig. 1A, GP-17 noticeably reduced the levels of intracellular Aβ₄₀ and Aβ₄₂ in APP695swe cells in a time-dependent manner (p < 0.01). A reduction in Aβ is primarily caused by decreased AβPP expression, autophagy enhancement, or proteases activation [17]. Thus, we desired to determine whether the reductions in Aβ involved changes in AβPP by measuring the expression of full-length AβPP in cell lysates. Immunoblotting experiments demonstrated a significant decline in the expression of full-length AβPP in cells incubated with GP-17 (Fig. 1B and 1 C, p < 0.01). To investigate whether the downregulation of AβPP was a consequence of decreased transcription, experiments were performed in the presence of actinomycin D (a transcriptional inhibitor). The result showed further reductions in AβPP levels in the presence of GP-17 (Fig. 1B, p < 0.01), which indicated that the effect of GP-17 on the removal of AβPP was not dependent on transcriptional regulation. We further investigated the possibility that GP-17 stimulated proteasome-dependent degradation pathway of AβPP. APP695swe cells were incubated with GP-17 in the presence or absence of the proteasome inhibitor MG132. The effect of GP-17 was partially blunted but was incompletely abrogated by MG132. A significant decrease in AβPP expression was observed in APP695swe cells after GP-17 treatment in combination with MG132 as compared with MG132 treatment alone (Fig. 1 C, p < 0.01). Therefore, proteasome is only partly responsible for the observed decline in GP-17-mediated AβPP degradation.

We explored the hypothesis that GP-17 favors AβPP degradation via the autophagy pathway. To determine whether GP-17 affected autophagy, control and APP695swe cells were treated with 10 μM GP-17 for 12 h, and autophagosome formation was investigated by transmission electron analysis. As illustrated in Fig. 1D, APP695swe overexpression significantly elevated the number of autophagosomes (p < 0.01). Unexpectedly, in GP-17-treated APP695swe cells, more autophagosomes were observed (p < 0.01). To confirm our findings, control and APP695swe cells transduced with pCMV-GFP-LC3 were incubated with GP-17 (10 μM) for 12 h. As illustrated in Fig. 2A and 2B, the overexpression of APP695swe significantly increased GFP-LC3 puncta as compared with control cells (p < 0.01). However, in GP-17-treated APP695swe cells, a noticeable increase in GFP-LC3 puncta was observed (p < 0.01). We further investigated the time-dependent changes in GFP-LC3 puncta by the incubation of control and APP695swe cells transduced with pCMV-GFP-LC3 with GP-17 for increasing periods of time (2, 4, 8, 12, 16, 20, and 24 h). High-content analysis showed that, in GP-17-treated APP695swe cells, the GFP-LC3 puncta/nucleus increased to the maximum by 1.5-fold after 12 h GP-17 treatment as compared with those of APP695swe (Fig. 2 C, p < 0.01). Thus, 10 μM and 12 h were chosen for the dose and treatment time of GP-17, respectively. The GFP-LC3 puncta/nucleus reduced gradually to a level below the baseline after 24 h of GP-17 treatment. However, the exposure of control cells to GP-17 for 24 h did not affect the number of GFP-LC3 puncta (Fig. 2 C, p > 0.05), which suggested that GP-17 might have no autophagy-inducing effect in control cells. According to the immunoblotting results, the overexpression of APP695swe remarkably increased the ratio of LC3-II/LC3-I as compared with the control (p < 0.01), but GP-17 increased the conversion of LC3-I into LC3-II in APP695swe cells (Fig. 2D, p < 0.01). To advance our understanding of the function of autophagy in the GP-17-mediated removal of AβPP, Atg5 (an essential Atg for autophagy induction) was silenced by Atg5 siRNA in APP695swe cells. The inhibitory efficacy of Atg5 siRNA on Atg5 expression was confirmed, and the GP-17-mediated augmentation of Atg5 expression was abolished by Atg5 siRNA in APP695swe cells (Fig. 2E, p < 0.01). As expected, Atg5 silencing abrogated the degradation of APP elicited by GP-17 (Fig. 2E, p < 0.01). Treatment with GP-17 also failed to decrease intracellular Aβ₄₀ and Aβ₄₂ levels in APP695swe cells transduced with Atg5 siRNA (Fig. 2F, p < 0.01).

GP-17 rescued autophagy flux through enhancing lysosome biogenesis in APP695swe cells

To verify that GP-17-mediated increase in LC3-II level represented increased autophagic flux rather than an accumulation of LC3-II, the level of the SQSTM1/p62 was quantified. APP695swe overexpression led to a significant increase in SQSTM1/p62 level, whereas the exposure of APP695swe cells with GP-17 resulted in a marked decrease in SQSTM1/p62 level (Fig. 2E, p < 0.01).

To confirm the effect of GP-17 on the autophagy flux, control and APP695swe cells were treated with GP-17 in the presence or absence of 3-MA or bafilomycin A1. Flow cytometry analysis revealed that the percentage of cells with endogenous saponin resistant LC3-II was significantly decreased in all 3-MA-treated group (Fig. 3A, p < 0.01). This finding suggested that APP695swe cells had functional autophagosome generation. Bafilomycin A1, an inhibitor of the vacuolar H+ ATPase, could impair lysosome acidification and could block the fusion of autophagosomes with lysosomes. Upon treatment with bafilomycin A1, the percentage of cells containing endogenous saponin resistant LC3-II was elevated in the control cells (Fig. 3A, p < 0.01) whereas unchanged in APP695swe cells (Fig. 3A, p > 0.05). This result suggested that the overexpression of APP695swe caused an impairment of autophagosome degradation. On the contrary, GP-17 treatment resulted in an elevation in the percentage of cells containing endogenous saponin resistant LC3-II in APP695swe cells in the presence of bafilomycin A1 (Fig. 3A, p < 0.01). This finding indicated an enhancement of the autophagy flux in GP-17-treated APP695swe cells.

An efficient autophagy process relies on the fusion of autophagosomes with lysosomes [4]. LAMP-1, a lysosomal structural protein, has a pivotal function in lysosome fusion [18]. To determine whether GP-17 influenced lysosome biogenesis, the level of LAMP-1 was monitored. The overexpression of APP695swe markedly decreased the level of LAMP-1 (Fig. 3B, F, p < 0.01), which was markedly alleviated following GP-17 treatment (Fig. 3B, F, p < 0.01), as detected by immunoblotting and immunofluorescence. Therefore, GP-17 stimulated lysosome biogenesis in APP695swe cells. The acidic pH of the lysosomal lumen plays a central role in lysosome-dependent autophagosome degradation. Accordingly, alteration in lysosomal pH was evaluated using LysoTracker Red DND-99. A remarkable reduction in the abundance of acidic organelles stained with LysoTracker Red was observed in APP695swe cells (Fig. 3C, D, p < 0.01). This phenomenon indicated that the acidic conditions of lysosome were disturbed in APP695swe cells. Conversely, the exposure of APP695swe cells to GP-17 led to a marked increase in LysoTracker Red staining (Fig. 3C, D, p < 0.01). This observation suggested that GP-17 re-acidified lysosomal pH in APP695swe cells. We further investigated the time-dependent changes in LysoTracker staining by the incubation of control and APP695swe cells with GP-17 for increasing periods of time (2, 4, 8, 12, 16, 20, and 24 h). At these time points, LysoTracker Red fluorescence intensity was detected by high-content analysis. Treatment with GP-17 for 12 h led to a fourfold increase in LysoTracker Red fluorescence intensity in APP695swe cells (Fig. 3E, p < 0.01), whereas LysoTracker Red fluorescence was progressively decreased to twofold of the baseline at 24 h in GP-17-treated APP695swe cells (Fig. 3E, p < 0.01). However, up to 24 h treatment with GP-17 did not affect LysoTracker staining in control cells (Fig. 3E, p > 0.05). Notably, the exposure of APP695swe cells to GP-17 promoted the delivery of autophagosomes to the lysosomes, as revealed by co-localization between LC3-II puncta and LAMP-1 stained vesicles(Fig. 3F, G, p < 0.01).

GP-17 stimulated autophagy and lysosome biogenesis through TFEB activation in APP695swe cells

TFEB has been implicated in the regulation of autophagy and lysosome biogenesis [19]. To explore whether TFEB was involved in GP-17-mediated autophagy induction and lysosome biogenesis, the sub-cellular localization of TFEB was monitored using confocal immunofluorescence microscopy. TFEB localized predominantly in the cytoplasm of control cells, and the exposure of control cells to GP-17 did not induce cytoplasm-to-nucleus shuttling of TFEB. However, GP-17 significantly increased the percentage of APP695swe cells with TFEB nuclear translocation in APP695swe cells (Fig. 4A, B, p < 0.01). As expected, similar results were obtained by the detection of protein expression of TFEB in nuclear fraction and total TFEB by immunoblotting. GP-17 significantly increased the protein expression of TFEB in nuclear fraction (Fig. 4C, p < 0.01) but did not affect that of total TFEB of APP695swe cells (Fig. 4C, p > 0.05). These results indicated that GP-17 elicited the nuclear translocation of TFEB in APP695swe cells. We also detected concomitant increases in PTEN expression (Fig. 4C, p < 0.01).

We considered that 14-3-3 plays a key regulatory role in nuclear transport of TFEB [9, 11]. To further understand the mechanism that regulates TFEB, the interaction between TFEB and 14-3-3 was detected by Co-IP experiments. The interaction between TFEB and 14-3-3 was detected in both control and APP695swe cells. GP-17 significantly reduced the amount of 14-3-3 co-immunoprecipitated by TFEB in APP695swe cells (Fig. 4D, p < 0.01) rather than in control cells (Fig. 4D, p > 0.05). This phenomenon suggested that GP-17 released TFEB from TFEB/14-3-3 complex in APP695swe cells.

These results prompted us to investigate the role of TFEB in GP-17-mediated autophagy activation, lysosome biogenesis, and AβPP degradation using TFEB siRNA. As shown in Fig. 4E, TFEB siRNA efficiently reduced the total TFEB expression (p < 0.01) and the nuclear localization of TFEB in APP695swe cells (p < 0.01). The increased PTEN and LAMP-1 expression induced by GP-17 did not occur when TFEB was silenced (p < 0.01). Notably, the increased LC3-II/LC3-I ratio and the reduced p62 expression in GP-17-treated APP695swe cells were abrogated by knocking down TFEB (p < 0.01). In summary, these results indicated that TFEB plays a central role in GP-17-stimulated autophagy and lysosome biogenesis. Notably, the reduction in AβPP expression and the removal of Aβ mediated by GP-17 were blunted by transfection with TFEB siRNA (Fig. 4E, F, p < 0.01). These phenomena supported that TFEB nuclear translocation is essential to mediate the effects of GP-17 on the removalof AβPP.

GP-17 enhanced autophagy and lysosome biogenesis via TFEB activation in APP/PS1 mice

A significant increase in nuclear TFEB level but an unchanged total TFEB were observed, which coincided with an elevation in PTEN and LAMP-1 expression in cortex and the hippocampus of APP/PS1 mice after GP-17 treatment as compared with those of APP/PS1 mice (Fig. 5A, and Supplementary Fig. 1A, p < 0.01). APP/PS1 mice showed elevated LC3-II/ LC3-I ratio in both the cortex and the hippocampus, as well as increased p62 expression as compared with those of WT mice (Fig. 5A, and Supplementary Fig. 1A, p < 0.01). This finding suggested that autophagy flux is impaired in the brain of APP/PS1 mice. A higher ratio of LC3-II/LC3-I but a marked reduction in p62 level were observed in both the cortex and the hippocampus of GP-17-treated APP/PS1 mice (Fig. 5A, and Supplementary Fig. 1A, p < 0.01). We further assessed the effect of GP-17 on AβPP expression and Aβ deposition in the brain of APP/PS1 mice. Immunofluorescence and immunoblotting results showed that APP/PS1 mice exhibited a noticeable increase in AβPP expression and Aβ plaques in both the cerebral cortex and the hippocampus. GP-17 markedly reduced the numbers of Aβ plaques in cerebral cortex and hippocampus of APP/PS1 mice (Fig. 5B, and Supplementary Fig. 1B, p < 0.01). This finding was validated by ELISA, as insoluble and soluble forms of Aβ₄₀ and Aβ₄₂ in the brain of APP/PS1 mice were reduced after GP-17 treatment (Fig. 5C, p < 0.01). In summary, these findings indicated that GP-17 enhanced autophagy flux and lysosome biogenesis through TFEB activation and markedly remove Aβ in the brain of APP/PS1 mice.

GP-17 recovered spatial learning and memory in APP/PS1 mice

Morris water maze tests were conducted after 60 d of GP-17 treatment. As indicated in Fig. 6A and B, all mice had a shorter latency to find the platform on day 5 of training than that on day 1. The vehicle-treated WT mice had better performance than vehicle-treated APP/PS1 mice from day 2 to day 5 (p < 0.01). This result suggested that APP/PS1 mice displayed obvious deficiency in spatial learning ability. The vehicle-treated APP/PS1 mice had a longer latency than that of GP-17 treated mice (p < 0.01). On day 6, the ratio of the time spent in the target quadrant to the total swimming time (60 s) was recorded. Compared with the vehicle-treated APP/PS1 mice, GP-17-treated APP/PS1 mice showed a longer staying time (Fig. 6C, p < 0.01) and more times of crossing (Fig. 6D, p < 0.01). This finding indicated that GP-17 prevented spatial learning impairment in APP/PS1 mice. However, GP-17 insignificantly affected spatial learning abilities in WT mice (Fig. 6, p > 0.01).

DISCUSSION

This study constitutes the first demonstration that treatment with GP-17 (40 mg/kg) for 60 d effectively restored the spatial learning and memory of the APP/PS1 mice without enhancing cognitive function in WT mice (Fig. 6). Notably, GP-17 significantly decreased soluble and insoluble fraction of Aβ in the brain of APP/PS1 mice, and markedly prevented the formation of Aβ plaques in the hippocampus and cortex of APP/PS1 mice (Fig. 5). To the best of our knowledge, this study is the first to uncover the therapeutic potentials of GP-17 in rodent models of AD. Importantly, treatment with GP-17 (10 μM) for 12 h caused a significant decline in the levels of full-length AβPP, Aβ₄₀, and Aβ₄₂ in APP695swe cells (Fig. 1). Furthermore, we focused on autophagy to investigate the mechanism underlying the GP-17-mediated removal of AβPP after the possibilities such as the changed AβPP transcription and an increased proteasomal degradation of AβPP were ruled out (Fig. 1).

Autophagic process is integrated and executed by various Atg proteins, including Atg8 (LC3) and Atg5 [20]. The electron microscopy study indicated that treatment with GP-17 led to an increase in the number of autophagosomes in APP695swe cells. GP-17 treatment could increase the conversion of LC3-I into LC3-II and could upregulate Atg5 expression, as determined by western blotting. GP-17 could elevate LC3-II puncta in APP695swe cells transduced with GFP-LC3 construct (Fig. 2). Notably, in the hippocampus and cortex of APP/PS1 mice, the ratio of LC3-II/LC3-I was also increased upon GP-17 treatment (Fig. 5). These results indicated that GP-17 had autophagy-inducing effects in cellular and rodent models of AD. As expected, the GP-17-mediated degradation of full-length AβPP and removal of Aβ₄₀ and Aβ₄₂ was prevented by the transfection of APP695swe cells with Atg5 siRNA (Fig. 2). This finding further substantiated the pivotal role of autophagy in the GP-17-mediated degradation of AβPP and clearance of Aβ.

Increased autophagosomes levels could stem from either overinduction of autophagy or dysfunctional autophagy flux. To distinguish these possibilities, control and APP695swe cells were treated with GP-17 with or without 3-MA and bafilomycin A1. 3-MA was used to prevent the formation of autophagosomes, and bafilomycin A1 was used to block the fusion of autophagosomes and lysosome. In this study, we found that defective autophagy flux initiated a compensatory increase in autophagosomes generation in APP695swe cells. Surprisingly, GP-17 increased autophagy initiation with better preserved autophagy flux in APP695swe cells (Fig. 3). Notably, GP-17 reversed the dysfunctional autophagy flux in cellular and rodent models of AD, which was evidenced by an increase in LC3-II level with a reduction of p62 expression in APP695swe cells (Fig. 2), and in hippocampus and cortex of APP/PS1 mice (Fig. 5). To the best of our knowledge, this study is the first to demonstrate that GP-17 rescued autophagy flux in cellular and rodent models of AD.

A block in autophagic flux was found as a result of the combination of increased autophagosome and lysosomal dysfunction in APP695swe cells (Fig. 3). Therefore, secluding Aβ by autophagosomes formation is not enough, but doing so could result in the dysregulation of autophagy flux if the cell fails to generate enough lysosomes to break down autophagosomes. An exciting finding of this study was that GP-17 noticeably induced autophagosome formation and assisted the fusion of autophagosomes with lysosomes. This study demonstrated that GP-17 upregulated LAMP-1 in APP695swe cells (Fig. 3) and in the hippocampus and cortex of APP/PS1 mice (Fig. 5). Notably, GP-17 treatment led to an elevation of lysosome abundance and an increase in co-localization between LC3-II puncta and LAMP-1-stained organelles in APP695swe cells (Fig. 3). GP-17 increased autophagosome formation and assisted the fusion of autophagosome with lysosome through restoring the acidic conditions of lysosome, accompanied by the restoration of autophagic flux (Fig. 3). Most importantly, the duration of lysosome biogenesis was properly regulated by GP-17 (Fig. 3). This property is significant because persistent and excessive lysosome formation would disturb the functional autophagy and would be detrimental to neurons. Despite these intriguing observations, further studies about the exact mechanism by which GP-17 keeps balance between the autophagosome generation and lysosome biogenesis should be conducted.

Accumulated evidence has revealed that TFEB is a master transcription factor that regulates the transcription of lysosomal and autophagic genes in a coordinated fashion [7 , 19]. We proposed that targeting nuclear localization of TFEB may prove a viable and efficacious strategy to eradicate Aβ and to provide beneficial effects on AD. Interestingly, TFEB was harnessed by GP-17 to rescue defective autophagy and impaired lysosome biogenesis in cellular and rodent models of AD. In this study, the interaction between TFEB with 14-3-3 resulted in the cytoplasmic retention of TFEB in APP695swe cells, and treatment with GP-17 elicited the dissociation of TFEB/14-3-3 complex and led to the mobilization of TFEB into the nucleus. In the nucleus, TFEB increased the expression of its targeted proteins such as LAMP-1 and PTEN (Fig. 4). Notably, GP-17 treatment resulted in the nuclear translocation of TFEB, which coincided with an increase in the expression of LAMP-1 and PTEN in hippocampus and cortex of APP/PS1 mice (Fig. 5). We further investigated whether TFEB was responsible for the regulation of autophagy and lysosome biogenesis mediated by GP-17 in APP695swe cells. In the present study, the knockdown of TFEB abolished TFEB nuclear translocation, lysosomal enhancement, and the increased expression of LAMP-1 and PTEN mediated by GP-17 in APP695swe cells (Fig. 4). These results provided strong support for the central role of TFEB in GP-17-mediated autophagy and lysosome biogenesis in APP695swe cells. GP-17-mediated the reduction in AβPP expression, and the elimination of Aβ was also blocked by TFEB silencing in APP695swe cells (Fig. 4). Notably, previous studies have proven that TFEB is regulated by several receptors such as estrogen receptor, androgen receptor, and glucocorticoid receptor [21]; kinases such as mTORC1 [11], RRAG GTPases [22], RANKL-PKCβ [23]; and signaling pathways such as phosphatidylinositol-3-kinase (PI3K)-Akt [24] and adenosine monophosphate-activated protein kinase [25]. PTEN could activate autophagy by reciprocally controlling PI3K-Akt-mTOR signaling [26]. Given that mTOR itself is a TFEB target [27], the following hypothesis is reasonable: a positive feedback loop by which mTOR is inactivated through TFEB-PTEN-Akt signaling, which, in turn, promotes TFEB activation through PTEN-Akt-mTOR signaling, might be involved in GP-17-mediated autophagy initiation. Identifying other upstream kinases or signaling pathways involved in GP-17-mediated TFEB nuclear translocation will be interesting. These findings might help to expand the current view on the mechanism underlying neuroprotective effects of natural medicine or traditional Chinese medicine.

In summary, GP-17 facilitated the autophagy-dependent removal of Aβ through TFEB activation in cellular and rodent models of AD, and it might be a promising candidate for the prevention of AD.

Footnotes

ACKNOWLEDGMENTS

This work was supported by the National Natural Science Foundation of China (No. 81503290); Special Scientific Research for Traditional Chinese Medicine of State Administration of Traditional Chinese Medicine of China (No. 201507004); the Major Scientific and Technological Special Project for “Significant New Drug Formulation” (No. 2012ZX09501001004); Program for Innovative Research Team in IMPLAD (Grant No. IT1301); Special Research Project for TCM (Grant No. 201507004).

Authors’ disclosures available online ().

Appendix

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