A Newly Synthesized Rhamnoside Derivative Alleviates Alzheimer's Amyloid-β-Induced Oxidative Stress, Mitochondrial Dysfunction, and Cell Senescence through Upregulating SIRT3

Oxidative stress-induced mitochondrial dysfunction and cell senescence are considered critical contributors to Alzheimer's disease (AD), and oxidant/antioxidant imbalance has been a therapeutic target in AD. SIRT3 is a mitochondrial protein regulating metabolic enzyme activity by deacetylation and its downregulation is associated with AD pathology. In the present study, we showed that a newly synthesized rhamnoside derivative PL171 inhibited the generation of reactive oxidant species (ROS) induced by amyloid-β42 oligomers (Aβ42O), major AD pathological proteins. Moreover, the reduction of mitochondrial membrane potential (MMP) and the impairment of mitochondrial oxygen consumption triggered by Aβ42O were also prevented by PL171. Further experiments demonstrated that PL171 reduced the acetylation of mitochondrial proteins, and particularly the acetylation of manganese superoxide dismutase (MnSOD) and oligomycin-sensitivity-conferring protein (OSCP), two mitochondrial SIRT3 substrates, was suppressed by PL171. Mechanism studies revealed that PL171 upregulated SIRT3 and its upstream peroxisome proliferator-activated receptor-γ coactivator 1α (PGC-1α) under basal and Aβ42O-treated conditions. The inhibition of SIRT3 activity could eliminate the protective effects of PL171. Further, long-term treatment with Aβ42O increased the number of senescent neuronal cell, which was also alleviated by PL171 in a SIRT3-dependent manner. Taken together, our results indicated that PL171 rescued Aβ42O-induced oxidative stress, mitochondrial dysfunction, and cell senescence via upregulating SIRT3 and might be a potential drug candidate against AD.


Introduction
The neuropathological hallmark of Alzheimer's disease (AD) is the deposition of extracellular amyloid plaques in the brain due to the imbalance in the production and clearance of amyloid-β (Aβ) [1], as well as intracellular neurofibrillary tangles [2], leading to the damage and death in neurons. Aβ secreted outside exists in different assembly states, and a series of evidences demonstrated that soluble Aβ oligomers are more pathogenic than larger, insoluble, highly aggregated fibril [3,4]. Mitochondria are dynamic organelles in eukaryotic cells playing a central role in ATP production, cellular calcium buffering, and apoptosis [5]. The reduction of mitochondria in its mass and function has emerged as another pathological feature in AD [6]. In recent years, some studies have shown that Aβ is imported into the mitochondria via the translocase of the outer membrane complex, providing a strong rationale that mitochondria also serve as targets for Aβ, contributing to cognitive decline and memory loss [7,8]. Aβ destroyed mitochondrial homeostasis and interfered with the enzymatic activity of the complex in the mitochondrial electron transport chain (ETC), resulting in the impairment of the mitochondrial membrane potential (MMP) [9,10]. And Aβ could cause serious oxidative damage by the overproduction of reactive oxidative species (ROS) and damage mitochondrial oxygen consumption directly leading to the reduction of ATP production [11].
Protein acetylation is a posttranslational process regulating global mitochondrial functions [12]. SIRT3 belongs to the sirtuin family and is located in the mitochondrial matrix, exhibiting a robust deacetylase activity [13,14]. It regulates the activity of mitochondrial metabolic enzymes, such as manganese superoxide dismutase (MnSOD) [15] and oligomycin-sensitivity-conferring protein (OSCP) [16], by deacetylation and thereby reducing the overproduction of ROS under oxidative stress-dependent conditions such as aging and neural degeneration [13]. It has been found that SIRT3 is downregulated in the brain of AD patients and analyzing SIRT3 level may contribute to AD diagnosis [17,18]. Therefore, promoting SIRT3 expression or function could be a promising therapeutic strategy for AD treatment. Rhamnose and rhamnoside have antioxidant effects [19,20], while whether they could prevent Aβ-induced neuron dysfunction is unknown. The present study demonstrated that a newly synthesized rhamnoside derivative PL171 attenuated Aβ 42 oligomer-(Aβ 42 O-) induced oxidative stress, mitochondrial dysfunction, and cell senescence by upregulating SIRT3mediated antioxidant effects, indicating PL171 can counteract Aβ 42 O defects via SIRT3.
Then, the β-L-rhamnopyranoside product (500 mg, 0.86 mmol) was dissolved in 8.0 mL of dry THF, and TBAF (1 M in THF, 0.86 mL, 0.86 mmol) was added dropwise to the solution at rt. The reaction mixture was stirred for 2 hours, after which it was diluted with THF 20 mL. The mixture was washed with brine and the combined organic layers were dried over MgSO 4 , and concentrated in vacuum. The residue was purified by chromatography on silica gel with eluent (petroleum ether-EtOAc, 5 : 1 to 1 : 1), and the phenol product 340 mg (0.73 mmol, 85%) was obtained.
The obtained phenol product 300 mg (0.65 mmol) was dissolved in 3.0 mL of CH 2 Cl 2 . 33% CH 3 NH 2 in CH 3 OH 0.5 mL was added to the solution at 0°C during 5 min. The resulting mixture was stirred for 1 hour at 0°C. The mixture was concentrated in vacuum and the resulting residue was purified by chromatography on silica gel with eluent (CH 2 Cl 2 -CH 3 OH, 20: 1 to 6: 1), and 140 mg (0. 41 [23][24][25]. Aβ 42 peptides were purchased from (Genic-Bio, A-42-T-2). Briefly, the hexafluoroisopropanol-(HFIP-) treated Aβ 42 peptides were resuspended in dimethyl sulfoxide (DMSO) and then diluted in DMEM/F12 phenol-red free medium to achieve a 100 μM concentration. The diluted Aβ 42 peptides were then vortexed for 15 s followed by incubation for 24 h at 4°C. The formation of Aβ 42 O were previously validated in our laboratory by dot blots, atomic force electromicroscopy, and western blot assays [23,26].
2.3. Cell Culture. SK-N-SH cells were purchased from ATCC. The cell line was cultured in Modified Eagle's Medium (MEM) with 10% fetal bovine serum (FBS) and 100 U/mL penicillin and 0.1 mg/mL streptomycin in a humidified incubator with 5% CO2/95% air (v/v) at 37°C.
2.4. Cell Viability. SK-N-SH cells were seeded in 96-well plate at 1 × 10 4 cells/well. After the treatment with PL171 for 24 h at indicated concentrations, cell viability was detected using Cell Titer-Glo Luminescent Assay (Promega, G7573), following the manufacturer's guidelines. The values were measured by BioTek SynergyNEO (BioTek, USA).

Mitochondria
Isolation. The cellular mitochondria were extracted following the manufacturer's instructions with some modifications (Beyotime, C3601). SK-N-SH cells (1:5 × 10 6 ) were seeded into 60 mm dishes. After the required treatments, cells were washed once with PBS, dissociated with trypsin-EDTA solution, and collected by centrifugation at 200 g for 10 min. The cell pellets were gently resuspended in PBS precooled in an ice bath followed by centrifugation at 600 g for 5 min at 4°C. The pellets were gently resuspended with 1 mL mitochondrial separation reagent supplemented with (100 μM) PMSF and then incubated on ice for 10 minutes. Cell suspensions were then homogenized on ice with a 1 cc insulin syringe 28G1/2, drawing through the 2 Oxidative Medicine and Cellular Longevity needle 10 times. After centrifugation at 600 g for 10 min at 4°C, the supernatants were collected and recentrifuged at 11,000 g for 10 min at 4°C to get the mitochondria. The mitochondria lysates were then used for western blot analysis.
2.11. Western Blot. Cells (1 × 10 5 cells/well) were treated with PL171 for 24 h or pretreated with PL171 for 4 h followed by Aβ 42 O treatment for another 24 h. For mitochondria lysate preparation, cells at 1:5 × 10 6 cells/well density were seeded and mitochondria were isolated as previously described. Total cell lysates or mitochondria lysates were separated by 10 or 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto nitrocellulose membranes (400 mA constant current, 2 h, 4°C). Membranes were blocked with 5% nonfat milk in TBS containing 0.1% Tween-20 for 1 h at room temperature (RT).  Hoechst. The data are presented as mean ± SEM, n ≥ 3 independent experiments, * p < 0:05, * * p < 0:01, * * * p < 0:001, and * * * * p < 0:0001, analyzed by one-way ANOVA followed by Bonferroni's test. 4 Oxidative Medicine and Cellular Longevity  2.12. Statistical Analysis. The data were analyzed by Prism 6.0 (GraphPad Software Inc., San Diego, CA). Unpaired Student's t-test (two-tailed) was applied for the comparisons of two datasets, and one-way or two-way analysis of variance (ANOVA) with Bonferroni's posttest was used where more than two datasets were compared. Statistical significance was accepted at p < 0:05.  (Figure 1(a)]. Thus, 10 μM of Aβ 42 O was applied for the subsequent experiments. We then tested whether PL171, as shown in the structure (Figure 1(b)), can modulate Aβ 42 Oinduced ROS promotion. Treatment with PL171 up to 30 μM for 24 h did not influence cell viability (Figure 1(c)). The effect of PL171 on the basal level of ROS was investigated.   each), which allowed the measurement of basal respiration (b), mitochondrial ATP production (c), and the maximal respiration (d). The data are presented as mean ± SEM, n = 3 independent experiments, * p < 0:05, analyzed by one-way ANOVA followed by Bonferroni's test. 6 Oxidative Medicine and Cellular Longevity  We observed that after 24 h treatment, PL171 decreased basal ROS production in a dosage-dependent manner with around 14% reduction made by 30 μM of PL171 (Figure 1(d)). Aβ 42 O (10 μM) consistently induced the increase of ROS which however was dose dependently reduced by the pretreatment with PL171 (Figures 1(e) and 1(f)). PL171 at 30 μM almost completely inhibited Aβ 42 O-induced ROS generation. To specifically detect the mitochondrial ROS, a mitochondrial superoxide indicator, MitoSOX, was applied. Data consistently showed that Aβ 42 O (10 μM, 24 h) stimulated mitochondrial ROS by about 26% which was significantly suppressed by the preincubation with PL171 (30 μM, 4 h) (Figure 1(g)). These results indicate that PL171 protects neuronal cells from Aβ 42 O-induced oxidative stress.

PL171 Prevented Aβ 42 O-Induced MMP Reduction in SK-N-SH Cells. Aβ 42 O can induce the loss of MMP.
In the present study, JC-1 probe was used to evaluate MMP in SK-N-SH cells. Red fluorescence and green fluorescence represented high and low mitochondrial membrane permeability, respectively, and the ratio could represent the change of MMP. Compared with the control group, treatment with Aβ 42 O largely enhanced green fluorescence intensity (Figure 2(a)) and significantly reduced the red/green fluorescence (Figure 2(b)), indicating MMP depolarization induced by Aβ 42 O. By contrast, Aβ 42-1 as the negative control had no obvious effect (Figures 2(a) and 2(b)). The effect of Aβ 42 (Fig. S4). Interestingly, pretreatment with PL171 for 4 h dose dependently prevented Aβ 42 O-impaired MMP (Figures 2(c) and 2(d)). Aβ 42 O (10 μM, 24 h) induced the reduction of MMP by 34% which was attenuated to around 10% by preincubation with 30 μM of PL171 for 4 h. This protective effect of PL171 was even more profound when extending the period of PL171 preincubation to 24 h (Figure 2(e)). And meanwhile, PL171 did not change MMP in the cells without Aβ 42 O while rotenone as a positive control produced around 37% reduction (Figure 2(f)).

PL171 Inhibited Aβ 42 O-Induced Reduction of Oxygen Consumption in SK-N-SH Cells. Previous results showed that
Aβ accumulated in the mitochondria, thus resulting in ATP depletion, decline of respiration rate, and low respiratory enzyme activity [10,27]. To further detect the effect of PL171 on mitochondrial function, we analyzed oxygen consumption rate (OCR) using a Seahorse instrument. In our study, compared to the control group, Aβ 42 O (10 μM, 24 h) impaired OCR, and however, the presence of PL171 (30 μM, 4 h pretreatment) inhibited Aβ 42 O-induced mitochondrial impairment (Figure 3(a)). Aβ 42 O declined basal respiration by 21% which was rescued to the control level by preincubation with 30 μM of PL171 for 4 h (Figure 3(b)). Meanwhile, Aβ 42 O reduced ATP production by about 25% while pretreatment with PL171 (30 μM) for 4 h restored the ATP level to the level similar as the control (Figure 3(c)). Compared with the control group, Aβ 42 O impaired the mitochondrial maximal respiration by 22% which was also prevented in the presence of PL171 completely (Figure 3(d)). Taken together, our data suggest that PL171 can inhibit Aβ 42 O-induced reduction of oxygen consumption, including ATP production, basal respiration, and maximal respiration and maintain healthy mitochondrial function.   Oxidative Medicine and Cellular Longevity

PL171 Promoted Mitochondrial SIRT3 Level and Its
Activity. Mitochondrial protein acetylation is tightly associated with mitochondrial function [28,29]. Firstly, we detected the effect of PL171 on the acetylation status of mitochondrial proteins. SK-N-SH cells were treated with various concentrations of PL171 for 24 h followed by mitochondria isolation. Data showed that PL171 reduced total acetylation of mitochondrial protein dose dependently (Figure 4(a)). To investigate the time course of mitochondrial protein deacetylation, cells were treated with PL171 at 30 μM for 0.5-24 h, and data presented that 24 h treatment produced maximum reduction of acetylation (Figure 4(b)). Since SIRT3 plays a significant role in mitochondrial protein deacetylation [30], the expression of SIRT3 in mitochondria was determined. The immunoblotting showed that PL171 increased mitochondrial SIRT3 by 36% (Figures 4(c) and 4(f)). Furthermore, we asked if the upregulation of SIRT3 promoted its activity for substrate deacetylation. The acetylation level of the SIRT3 substrates, manganese superoxide dismutase (SOD2) and oligomycin-sensitivity-conferring protein (OSCP), was detected using antibodies that specifically detect MnSOD acetylation at K-68 and OSCP acetylation at K-139 by immunoblotting. PL171 decreased the acetylation of MnSOD and OSCP in a dose-dependent manner and 30 μM of PL171 reduced acetylation of MnSOD (SODk68/MnSOD) and OSCP (ATP5O/OSCP) by about 20% and 36%, respectively (Figures 4(c)-4(e)). However, treatment with a SIRT3 inhibitor (3-TYP, 20 μM) significantly blocked the effect of PL171 (Figures 4(g)-4(h)). Furthermore, Aβ 42 O (10 μM) increased the acetylation level of MnSOD which was significantly downregulated by preincubation with 30 μM of PL171 for 4 h (Figures 4(j) and 4(k)).  , 24 h). The protein expression of AMPK phosphorylation (pAMPK), total AMPK, and SIRT3 was detected by western blotting. Actin was used as a loading control. The relative pAMPK and SIRT3 were quantified (h, i). (j) After preincubation with PL171 as indicated, cells were treated with 10 μM Aβ 42 O for 24 h, and then cell lysates were prepared and analyzed using western blotting against SIRT3 and PGC-1α antibody. (k, l) The quantifications of relative SIRT3 and PGC-1α protein level in (j). The data are presented as mean ± SEM, n ≥ 3 independent experiments, * p < 0:05, * * p < 0:01, analyzed by one-way or two-way ANOVA followed by Bonferroni's test. 10 Oxidative Medicine and Cellular Longevity All these demonstrate that PL171 can protect mitochondrial function by facilitating mitochondrial protein deacetylation through promoting SIRT3 function.  (Figs. S5D-F). The expression of the SIRT3 gene is shown to be controlled by the transcription factor PGC-1α which can be regulated by AMPactivated protein kinase (AMPK) signal pathway [31,32].

Discussion
In AD, mitochondrial dysfunction could be comprised of three different aspects: (1) mitochondrial dynamic or morphology, (2) bioenergetics (ATP and oxidative stress), and (3) transport [34]. Regarding bioenergetics defects of mitochondria in AD, many patients and disease models display reduced ATP production, excessive ROS generation, and significant respiratory defects [10]. Moreover, AD pathological proteins including Aβ and tau have been demonstrated to impair mitochondrial mass and function [35,36]. Although it is unclear whether mitochondrial dysfunction comes  . The data are presented as mean ± SEM, n ≥ 3 independent experiments, * p < 0:05, * * * p < 0:001, analyzed by one-way ANOVA followed by Bonferroni's test. 12 Oxidative Medicine and Cellular Longevity earlier than the appearance of pathological proteins or not, all these studies emphasize the essential roles of mitochondria in AD pathogenesis and targeting mitochondria dysfunction could be beneficial for disease treatment. Indeed, a variety of antioxidants such as resveratrol [37,38], curcumin [39], and idebenone have been shown to improve memory deficit in AD [40,41]. In the present study, we demonstrate that a newly designed natural compound derivative PL171 may have antioxidant effects and prevent Aβ-induced mitochondrial dysfunction in human neuronal cells, indicating that PL171 could be a therapeutic agent for AD by targeting the mitochondria. The mechanism of Aβ-mediated mitochondrial dysfunction is not exactly clear yet. In recent years, some groups have explored relevant mechanisms to impact mitochondrial function in AD. SIRT3 is the main mitochondrial sirtuin involved in protecting stress-induced mitochondrial integrity and energy metabolism and is highly associated with the pathogenesis of AD [13]. In the cortex of APP/PS1 double transgenic mice which are overproducing Aβ, both the mRNA and protein levels of SIRT3 are declined [42] and literature shows a negative association between SIRT3 expression and Aβ level in AD patients [17]. Thus, SIRT3 has been suggested as a molecular target for treating aging and age-related diseases [43,44]. Here, we observed that Aβ 42 O induced the reduction of SIRT3 expression and its activity, further proving that SIRT3 is involved in Aβ-mediated mitochondrial dysfunction. The prevention of SIRT3 reduction by PL171 attenuated Aβ 42 O-induced neuronal defects, which were abolished by the SIRT3 inhibitor, suggesting that SIRT3 could be a therapeutic target for AD treatment.
PGC-1α, a transcriptional coactivator for the peroxisome proliferator-activated receptor-γ (PPARγ) and for other transcription factors is involved in the regulation of oxidative phosphorylation, lipid metabolism, and mitochondrial biogenesis [45]. PGC-1α has protective effects against AD pathology. For example, PGC-1α has been reported to downregulate the transcription and expression of BACE1, which  . The data are presented as mean ± SEM, n ≥ 3 independent experiments, * * * p < 0:001 and * * * * p < 0:0001, analyzed by one-way ANOVA followed by Bonferroni's test.
13 Oxidative Medicine and Cellular Longevity results in reduced Aβ generation and increased nonamyloidogenic sAPPα levels [46]. Notably, it was revealed that PGC-1α was decreased in the brain of AD patients and the content of PGC-1α protein was negatively correlated with Aβ levels [33]. Furthermore, in vitro studies demonstrated that Aβ reduced PGC-1α expression and PGC-1α could restore Aβ neurotoxicity [47,48]. Here, PL171 significantly increased both mRNA and protein levels of PGC-1α and prevented Aβ-induced decline of protein. It has been reported that AMPK activation can stimulate CREB-mediated PGC-1α expression which regulates ERRα binding to the motif in SIRT3 promoter and promotes SIRT3 gene level [49,50]. We observed that PL171 stimulated AMPK activation and its inhibition abolished the effect of PL171 on SIRT3. Thus, we suspect that PL171 may improve mitochondrial function via AMPK/PGC-1α/SIRT3 axis.
Cell senescence is a biological process that involves several key elements including mitochondrial dysfunction, ROS production, inflammation, and DNA damage and plays a key role in promoting aging and age-related diseases, such as AD [51,52]. Together with previous studies [53,54], our data show that long-term treatment with Aβ 42 O facilitate the number of senescent neuronal cells properly by stimulating ROS generation and mitochondrial dysfunction. Selective elimination of senescent cells or inhibition of cell senescence process is now considered a promising strategy for the treatment of age-associated disorders [55]. Rhamnose and rhamnoside have antioxidant effects and show benefits on skin aging [19,20], while whether they could influence Aβ 42 Oinduced neuronal senescence is unknown. Here, as a designed rhamnoside derivative, PL171 can not only prevent Aβ 42 O-induced oxidative stress and mitochondrial impairment but also inhibited Aβ 42 O-mediated cell senescence. All these effects were absent when the activity of SIRT3 was blocked, indicating that PL171 has antiaging or anti-AD effects via targeting SIRT3. Notably, all these effects of PL171 were examined in a cell line which is deficient for in vivo interoperation. In the future, more relevant in vivo models should be applied to further investigate the therapeutic potential of PL171 on aging or AD intervention.

Conclusion
PL171 can counteract Aβ-induced oxidative stress-mediated mitochondrial dysfunction and cell senescence via promoting SIRT3 function in human neuronal cells.

Data Availability
All data used to support the findings of this study are included within the article.

Conflicts of Interest
PX is a full-time employee of Shanghai EW Medicine Co., Ltd. The remaining authors declare no competing financial interests.