Ni Wang,Junyan Yang,Ruijun Chen,Yunyun Liu,Shunjie Liu,Yining Pan,Qingfeng Lei,Yuzhou Wang,Lu He,Youqiang Song,Zhong Li
Ginsenoside Rg1 ameliorates Alzheimer's disease pathology via restoring mitophagy
Alzheimer's disease,ginseng,ginsenoside Rg1,mitophagy,Parkin
Background Alzheimer's disease (AD) is a common form of dementia, and impaired mitophagy is a hallmark of AD. Mitophagy is mitochondrial-specific autophagy. Ginsenosides from Ginseng involve in autophagy in cancer. Ginsenoside Rg1 (Rg1 hereafter), a single compound of Ginseng, has neuroprotective effects on AD. However, few studies have reported whether Rg1 can ameliorate AD pathology by regulating mitophagy. Methods Human SH-SY5Y cell and a 5XFAD mouse model were used to investigate the effects of Rg1. Rg1 (1μM) was added to β-amyloid oligomer (AβO)-induced or APPswe-overexpressed cell models for 24 hours. 5XFAD mouse models were intraperitoneally injected with Rg1 (10 mg/kg/d) for 30 days. Expression levels of mitophagy-related markers were analyzed by western blot and immunofluorescent staining. Cognitive function was assessed by Morris water maze. Mitophagic events were observed using transmission electron microscopy, western blot, and immunofluorescent staining from mouse hippocampus. The activation of the PINK1/Parkin pathway was examined using an immunoprecipitation assay. Results Rg1 could restore mitophagy and ameliorate memory deficits in the AD cellular and/or mouse model through the PINK1-Parkin pathway. Moreover, Rg1 might induce microglial phagocytosis to reduce β-amyloid (Aβ) deposits in the hippocampus of AD mice. Conclusion Our studies demonstrate the neuroprotective mechanism of ginsenoside Rg1 in AD models. Rg1 induces PINK-Parkin mediated mitophagy and ameliorates memory deficits in 5XFAD mouse models.
Ginsenoside Rg1 ameliorates Alzheimer's disease pathology via restoring mitophagy

Alzheimer's disease (AD) is a common form of dementia, and impaired mitophagy is a hallmark of AD. Mitophagy is mitochondrial-specific autophagy. Ginsenosides from Ginseng involve in autophagy in cancer. Ginsenoside Rg1 (Rg1 hereafter), a single compound of Ginseng, has neuroprotective effects on AD. However, few studies have reported whether Rg1 can ameliorate AD pathology by regulating mitophagy.

Human SH-SY5Y cell and a 5XFAD mouse model were used to investigate the effects of Rg1. Rg1 (1μM) was added to β-amyloid oligomer (AβO)-induced or APPswe-overexpressed cell models for 24 hours. 5XFAD mouse models were intraperitoneally injected with Rg1 (10 mg/kg/d) for 30 days. Expression levels of mitophagy-related markers were analyzed by western blot and immunofluorescent staining. Cognitive function was assessed by Morris water maze. Mitophagic events were observed using transmission electron microscopy, western blot, and immunofluorescent staining from mouse hippocampus. The activation of the PINK1/Parkin pathway was examined using an immunoprecipitation assay.

Rg1 could restore mitophagy and ameliorate memory deficits in the AD cellular and/or mouse model through the PINK1-Parkin pathway. Moreover, Rg1 might induce microglial phagocytosis to reduce β-amyloid (Aβ) deposits in the hippocampus of AD mice.

Our studies demonstrate the neuroprotective mechanism of ginsenoside Rg1 in AD models. Rg1 induces PINK-Parkin mediated mitophagy and ameliorates memory deficits in 5XFAD mouse models.

Alzheimer's disease (AD) is a common form of dementia. In 2019, the number of dementia patients was over 50 million, and this number is expected to reach 152 million by 2050 [1]. The typical hallmarks of AD patients' brains are β amyloid (Aβ) deposition and neurofibrillary tangles (NFTs). Also, the pathological changes, including neuronal loss [2], synaptic dysfunction [3], oxidative stress [4], and mitochondrial dysfunction [5], lead to memory loss and cognitive impairment of AD [6]. Mitochondrial dysfunction is associated with neurodegenerative diseases, such as AD and Parkinson's disease (PD) [7,8]. Fang et al demonstrated impaired mitophagy (mitochondrial-specific autophagy) in AD mouse models, neurons derived from AD patients' induced pluripotent stem cells (iPSC), and postmortem hippocampus of AD patients [5]. Mitochondria, as the powerhouse, generate ATP to support normal functions of almost all the cellular processes [9]. Mitophagy removes the defective or damaged mitochondria through several pathways [[10], [11], [12], [13], [14]]. The PINK1-Parkin signaling is widely studied in neurodegenerative diseases, including AD [10,11]. Upon dysfunctional mitochondria, PINK1 relocates onto the outer mitochondria membrane (OMM) and initiates mitophagy by adding phosphoryl groups to ubiquitin [10]. Parkin, an E3 ubiquitin ligase, is subsequently recruited to help to recognize the damaged mitochondria for sequestering [10,11]. Besides, other OMM receptors such as FUN14 domain-containing protein 1 (Fundc1), Bcl-2 interacting protein-3 like (BINP3L), and Bcl-2-like protein 13 (Bcl2L13) mediate mitophagy [[12], [13], [14]]. Fundc1 induces the hypoxia-associated mitophagy [12]. BINP3L, a member of the Bcl-2 family, initiates oxygen deprivation-related mitophagy in erythroid cells [13]. Bcl2L13, another Bcl-2 family member, mediates mitophagy through the Parkin-independent way [14]. Ginseng is regarded as the herbal supplement to provide “cellular energy” in traditional Chinese medicine (TCM). Recently, herbal medicines have been used in research and clinical studies for behavioral and psychological symptoms treatment associated with AD and other types of dementia [15]. Ginsenosides have protective effects on neurodegenerative diseases such as PD and AD [16,17]. Ginsenosides also play a role in autophagy and ER stress in cancer [18], but the molecular mechanism of them is not well-known. Ginsenoside Rg1 (Rg 1 hereafter), a single compound of Ginseng, is reported to reduce Aβ deposits in the AD mouse model [19]. Since ginsenosides are involved in autophagy and Rg1 plays a role in Aβ reduction. Hence, we hypothesized and demonstrated that Rg1 exerts its neuroprotective roles on AD models by restoring mitophagy.

Antibodies used in this study were summarized in supplementary Table 1. The Flag-tagged or GFP-tagged lentiviral plasmid encoding APPswe (Swedish) mutation is a kind gift from Dr. Zhang Zhigang (backbone plasmid, #17454). Ginsenoside Rg1 was purchased from Sigma Aldrich Company (22427-39-0) and dissolved in dimethyl sulfoxide (DMSO, Sigma, D8418) to the concentration of 10 mM for storage at −80°C. Aβ oligomer (AβO) was purchased from rPeptide company (A-1002-2) and diluted to the concentration of 100 μM for storage.

SH-SY5Y cells were maintained in DMEM/F12 (10% FBS, Gibco, USA) and cultured in a 5% CO2 incubator at 37°C. SH-SY5Y cells overexpressed Flag-tagged or GFP-tagged APPswe were generated by transfection using lipofectamine 3000 (Thermo Scientific, L3000015) and selected by 3μg/ml puromycin (InvivoGen, ant-pr-1). For drug treatments, SH-SY5Y cells were treated with 1 μM AβO with or without Rg1 (1μM) for the 24-hour and the equal volume of DMSO was used as controls. Cell lysates were harvested using RIPA lysis for the following experiments.

To examine the suitable concentration of Rg1, cell viability was tested by MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide) assay (Invitrogen, M6494). SH-SY5Y cells were seeded into a 96-well plate at a density of 1 × 104 cells/ml followed by adding different concentrations of Rg1 for 24 h. MTT working solution (5 mg/ml) was added to each well and incubated at 37°C for 4 hours. The medium was removed and the formazan crystals were dissolved in 200 μl of DMSO. The absorbance was determined by a spectrophotometer at 570 nm.

Experimental animals were maintained in a 12-hour light-dark cycle at 25°C according to the protocol of the Committee on the Use of Live Animals in Teaching and Research (CULATR), The University of Hong Kong. 5XFAD mice, expressing human APP mutations (K670N/M671L, I716V, and V717I) and PSEN1 mutations (M146L and L286V), were on C57BL/6 genetic background. Wild type C57BL/6 mice were used as controls. The mice (5-month-old) were divided into three groups: wild-type mice with saline, 5XFAD with saline, and 5XFAD with Rg1. The 5XFAD experimental mice were subjected to intraperitoneal injection at a daily dose of 10 mg/kg of Rg1 (dissolved in saline) for 30 consecutive days. The wild-type mice and half the number of 5XFAD were given the equal volume of saline as controls. Each mouse was subjected to a behavior test and then was sacrificed. Mice were perfused using 0.9% saline. The brains, livers and kidneys were dissected out for further experiments.

A circular pool (115 cm in diameter, 50 cm in height) was filled up with water to 30 cm in height. The tank was divided into quadrants. A 10-cm-in-diameter platform was placed at the center of the southeast quadrant as the target quadrant. Four clues were placed on the sidewalls surrounding the tank, which are visible for mice's spatial orientation. The swimming paths of mice were recorded by a video camera above the center of the pool. Each mouse was trained four times (60 s each time) from a different point for five consecutive days. The platform was removed on the sixth day and each mouse was allowed to swim for 60 s. The swimming path was captured by the camera and swim time in the target quadrant was analyzed using ImageJ software.

To examine whether Rg1 is safe for liver or kidney metabolism. Mouse liver or kidney was fixed in 4% paraformaldehyde (PFA) and embedded in paraffin. Tissue blocks were sectioned in 6 μm and stained with hematoxylin and eosin. Representative images were captured by the Olympus BX51 microscope.

For cultured cells, cells were seeded onto a 24-well plate containing coverslips followed by treating with Rg1 (1 μM) or DMSO for 24-hour. The medium was removed, and cells were fixed in 4% PFA at room temperature (R.T.) for 20 min. After washing in PBS, cells were permeated with 0.5% Triton X-100 for 5 min and blocked in 5% bovine serum albumin (BSA, Sigma-Aldrich, SRE0096) at R.T. for 1 hour. Cells were incubated in primary antibodies (supplementary Table 1) at 4°C overnight, incubated with secondary antibodies coupled to Alexa Fluor 488 or 568 (supplementary Table 1) at R.T. for 1 hour, and mounted in DAPI mounting medium (Vector Laboratories, H-1200). Representative immunofluorescent images were captured using an Olympus BX53 fluorescence microscope. For mouse tissue sections, mouse brains were fixed in 4% PFA at 4°C overnight, dehydrated in 30% sucrose, and embedded in O·C.T. medium. Blocks were sectioned at 10 μm and sections were washed in PBS. Tissues were permeated with 0.5% Triton X-100 at R.T. for 5 min. Samples were blocked in 10% horse serum (Thermo Scientific, 26050070) and 3% BSA at R.T. for 1 hour and incubated in primary antibodies (supplementary Table 1) at 4°C overnight, incubated with secondary antibodies at R.T. for 1 hour, and mounted with DAPI mounting medium. Representative fluorescent images were captured using Olympus BX53 fluorescence microscope or Zeiss LSM800 confocal microscope.

Lysates extracted from cultured cells and mouse tissues were precleared by protein A/G agarose beads (Santa Cruz Biotechnology, sc-2003) at 4°C for 30 min. After spinning at 13,500 rpm 4°C for 10 min, the supernatants were transferred into new tubes followed by incubation with anti-LC3B antibody and protein A/G agarose with gentle rocking at 4°C overnight. Beads were washed five times using cold cell lysis buffer (20 mM Tris-HCl, 150 mM NaCl, and 1% Nonidet P-40) and centrifuged at 3,000 rpm 4°C for 1 min. The pellets were re-suspended in sample loading buffer and heated at 100°C. Samples were stored at −80°C for the following analysis.

Cultured cells and mouse tissues were lysed in the lysis buffer with protease inhibitors (Roche, 11697498001) and phosphatase inhibitor (Roche, 04906845001). Protein samples were loaded onto 8%, 10%, or 15% Tris-Glycine gels and transferred to polyvinylidene difluoride (PVDF, Millipore, IPVH00010) membranes followed by blocking in 5% skim milk or 5% BSA. Membranes were incubated with primary antibodies (supplementary Table 1) at 4°C overnight. After washing, membranes were incubated with HRP-conjugated mouse or rabbit secondary antibodies (supplementary Table 1) at R.T. for 1 hour. Substrate (Thermo Scientific, 34096) was used to detect the signals and bands were quantified using Image J software.

Mouse hippocampus were isolated and fixed in 2.5% glutaraldehyde (Sigma-Aldrich, G6257) at 4°C overnight. The samples were washed in 0.1M sodium cacodylate buffer and post-fixed in 1% OsO4 at R.T. for 2 hours. The tissues were dehydrated in ethanol with increasing concentrations, followed by infiltration in epoxy resin/propylene oxide (1:1) at 37°C overnight. Tissues were embedded in epoxy resin at 60°C overnight. Ultra-thin sections were cut with a diamond knife (Diatome) and placed onto copper grids followed by staining with silver citrate. Images were captured by a Philips CM100 transmission electron microscope.

Statistical analysis was performed by Prism 7.0 software. All experimental data were subjected to the Kolmogorov-Smirnov test for normal distribution and Levene's test of Equal Variances for variance homogeneity. Continuous variables were shown as the mean ± SEM. Two-tailed student's t tests were analyzed to determine if the difference between two sets of data is significant. P values < 0.05 were considered as statistically significant and exact p values were provided in figures.

To investigate whether Rg1 can improve mitophagy in AD, we generated an AD cell model induced by β-amyloid oligomer (AβO) in the culture medium with two different concentrations (0.5 and 1μM). We chose to add 1μM of AβO in the human SH-SY5Y cells to mimic AD molecular changes. 1μM of AβO upregulated APP expression and inhibited autophagy/mitophagy (Fig. 1A–C and supplementary Figure 1A). To test the suitable concentration of Rg1, we examined the viability of SH-SY5Y cells in different concentrations using the MTT assay (Fig. 1D and E). A study has treated their experimental cells using 5μM of Rg1 [20]. However, we identified a better cell activity using1μM of Rg1 (Fig. 1E). To test whether Rg1 can ameliorate AβO-induced pathogenesis, we examined autophagy/mitophagy-associated markers. The results showed that Rg1 inhibits the phosphorylation of mTOR (Ser 2448) and ULK1 (Ser 757), which may initiate autophagy/mitophagy (Fig. 1F–H and supplementary Figure 1A). Another critical process of damaged mitochondria degradation is lysosome fusion with mitophagosome. Thus, we examined whether Rg1 can promote the lysosome fusion process. We found that Lamp1 (a lysosome marker) expresses in AβO-induced cells (Fig. 1I and J). The Lamp1-positive cells expressed LC3B-an autophagic marker (Fig. 1I). The Lamp1-positive cells express TOM20-an OMM marker (Fig. 1J), which indicates lysosome fusion with mitophagosome. We then investigated whether Rg1 can also exert its neuroprotective role in APPswe mutation-induced cell models. We generated two AD cell models that stably expressed Flag-tagged APPswe (Swedish) and GFP-fused APPswe (Fig. 2A and B, and supplementary Figure 1C). Similar to the findings in AβO-induced cells, we identified that Rg1 inhibits the phosphorylation of mTOR Ser2448 and ULK1 Ser757, which is a suppressor of autophagy (Fig. 2C and supplementary Figure 1C). In addition, Rg1 could regulate other mitophagy-related molecules via downregulating p62 and upregulating OPTN and LC3B (Fig. 2C, 2D, and supplementary Figure 1C).

We demonstrated that Rg1 restores mitophagy in vitro and further examined its neuroprotective effects in vivo. We employed the 5XFAD transgenic mouse, which was introduced with human APP with Swedish, London, and Florida mutations and two mutations (M146L and L286V) in the human PSEN1 gene [21]. The 5XFAD mice (5-month-age) were injected with Rg1 (10 mg/kg/d) (Fig. 3A), according to a previous study [22]. After 30 days, we performed Morris water maze tests and observed that the cognitive behavior of AD mice with saline was significantly worse than that of wild type mice (P = 0.017, Fig. 3C). But we found that Rg1-treated AD mice significantly spent more time in the target quadrant (vs. saline-treated AD mice, Fig. 3B and C). Although Rg1 benefits the liver and has renal protection effects [23,24], the side effects on the liver and kidney still need to be investigated. Then, we examined whether Rg1 is harmful to these organs, and we found no evident difference in the morphology of livers and kidneys between saline-treated and Rg1-treated AD groups (Fig. 3D). Rg1-treated hippocampus expressed a significantly decreased phosphorylation level of AMPK (Fig. 4A). This finding may suggest a relatively healthy mitochondria condition. Research has reported that stress of mitochondria or damaged mitochondria in AD may affect mitophagy and trigger cell death, such as apoptosis [25]. We detected a statistically significant decrease in expression levels of cleaved caspase 9 in the hippocampus of Rg1-treated AD (Fig. 4A). Besides, we observed significantly increased levels of LC3B (Fig. 4A and supplementary Figure 2A), indicating the activation of mitophagy in the hippocampus. We used the transmission electron microscope (TEM) to investigate mitophagic events in the hippocampus. We noticed that some cells underwent apoptosis and found many damaged mitochondria without phagophores in AD mice (blue arrow, Fig. 4B). However, we observed more mitophagosome formed in the hippocampus of Rg1-treated AD mice (red arrows, Fig. 4B) compared to saline-treated AD mice (blue arrow, Fig. 4B). Taken together, Rg1 may restore mitophagy and prevent cellular apoptosis in the AD mouse model.

The phosphorylation levels of mTOR (Ser 2448) and ULK1 (Ser 757) were inhibited in the hippocampus of the Rg1-treated AD mouse model. These findings suggest that Rg1 may be involved in autophagic initiation, indicated by the upregulation of VPS15. Mitophagy is a mitochondria-specific form of autophagy. It can be mediated by the PINK1-Parkin pathway or Parkin-independent receptors, which are localized on the OMM. To understand the regulatory signaling(s) underlying the neuroprotective roles of Rg1, we examined mitophagy-associated markers. We found that the PINK1-Parkin signaling should be involved in Rg1-induced mitophagy. Significant upregulation of OTPN (Fig. 5A and supplementary Figure 2B) was investigated, with minimal changes of receptors-Bcl2L13, BINP3L, and Fundc1 (Fig. 5A and C input and supplementary Figure 2B). Mitophagy is the removal process of damaged mitochondria. Phagophores with LC3B engulf the dysfuntional mitochondria to form into the mitophagosome. Immunoprecipitation with LC3B showed that more mitochondria (TOM20) bind to the phagophore (LC3B) in the Rg1-treated groups. This finding indicates that more mitochondria were enveloped in the phagophore (Fig. 5B and C). The result also demonstrated that Parkin or Fundc1 strongly binds to LC3B in the AβO-induced SH-SY5Y cells treated with Rg1 (Fig. 5B). However, we only observed a relatively strong binding affinity between Parkin and LC3B rather than between Fundc1 and LC3B (Fig. 5C) in the hippocampus of the Rg1-treated AD mouse model. This result may indicate a slight difference in regulatory pathways between human and mouse species. Our findings suggest that Rg1 may exert neuroprotective effects on the AD mouse model via the PINK1-Parkin signaling, which results in the degradation of the impaired mitochondria (Fig. 5D).

Cheng et al reported that APP overexpression is associated with neuronal cell apoptosis [26]. Since less apoptosis was detected in the Rg1-treated hippocampus (Fig. 4A). We examined the expression levels of APP in the AD cellular and mouse model. The results showed that Rg1 reduces APP expression in both AβO-induced cells (Fig. 6A and D and supplementary Figure 3A) and the 5XFAD mouse hippocampus (Fig. 6B and E and supplementary Figure 3A). This finding may also contribute to the inhibition of neuronal cell death. Moreover, the excessive accumulation of Aβ deposits causes cognitive defects in AD patients and AD models [5,27]. Many studies have reported that potential drugs could reduce the secretion of Aβ to alleviate cognitive impairment [5]. We then examined whether Rg1 could reduce Aβ accumulation. The size of Aβ deposits indicated by 6E10 (Fig. 6C) was significantly decreased (P < 0.001, Fig. 6F). This result indicates that Aβ deposits may be eliminated by the immune cells. Double-staining of Iba1 (a microglia marker) and Aβ plaque (6E10) showed the co-localization of microglia and Aβ deposits in the hippocampus of the Rg1-treated AD mouse model (Fig. 6G and Supplementary Figure 3B), which may indicate microglial phagocytosis.

Herbal medicine has been employed in recent research and clinical studies due to the neuroprotective effect [15]. Ginseng is prescribed in traditional Chinese medicine (TCM) to alleviate symptoms associated with memory deterioration [28]. Over 40 ginsenosides have been isolated from ginseng [29]. Ginsenoside Rg1 and Rb1 (active compounds of ginseng) could prevent neuronal death and improve cognitive deficiency in animals [30,31]. Rg1 and Rb1 could be absorbed in the GI tract after oral administration and cross the blood-brain barrier to exert their neuroprotective role in rats [32]. They reported that the bioavailability of Rg1 examined by Reversed Phase HPLC is relatively low (only 18.4%) 1 hour after oral administration [32]. Another study has intraperitoneally injected mAPP transgenic mice with 10 mg/kg of Rg1 [33]. Fang et al demonstrated that Rg1 improves cognition, reduces amyloid pathology, and regulates PKA/CREB pathway. In this study, we injected 5XFAD mouse model with Rg1 (10 mg/kg) for 30 days.

Mounting evidence suggests mitochondrial dysfunction is the hallmark of AD patients' brains [5]. Dysfunctional mitochondria fail to generate enough ATP to support cellular processes. Under low energy conditions, the phosphorylated AMPK (a conserved energy-sensing kinase) inhibits the mTOR (a serine/threonine-protein kinase) activity and initiates autophagy by activating ULK1 [34,35]. The active mTOR inactivates ULK1 through phosphorylating ULK1 at Ser757 [34]. The inhibition of mTOR often results from the phosphorylation of AMPK. However, we identified that the phosphorylation levels of mTOR and AMPK are both inhibited in this study. Two reasons may explain this conflict. First, Rg1 may facilitate healthy mitochondria to generate enough ATP to maintain normal physiologic function so that the phosphorylation of AMPK is not enhanced. Second, Rg1 cannot remove all the dysfunctional mitochondria in AD mice. It may result in partial inhibition of AMPK phosphorylation. Meanwhile, the physiopathological changes of AD are chronic processes. Short-term administration of Rg1 may not totally reverse AD pathology. However, the removal of dysfunctional mitochondria is still necessary to prevent cell death. Indeed, we investigated the inhibited phosphorylation of mTOR (Ser2448) and ULK1 (Ser757), which activates autophagy or mitophagy. Moreover, we detected the upregulation of mitophagy-associated molecules (e.g., OPTN and Parkin) in both AD cellular and mouse model. These findings indicate that Rg1 may exert the neuroprotective role via restoring mitophagy.

Several pathways mediate mitophagy, including PINK1-Parkin signaling and other Parkin-independent receptor-mediated pathways such as BINP3L, Fundc1, and Bcl2L13 [[12], [13], [14],36]. Upon the dysfunctional mitochondria accumulate, PINK1 recruits and actives Parkin on the OMM, which mediates the poly-ubiquitination of OMM proteins together with some autophagic adaptor molecules, including OPTN and LC3 family [37,38]. LC3B is recruited onto the phagophore, which engulfs the damaged mitochondria into the mitophagosome. Subsequently, lysosome fuses with the mitophagosome for degradation. Using immunoprecipitation with LC3B, we found a strong binding affinity between Parkin or TOM20 (an OMM marker) and LC3B in both Rg1-treated AD cellular and mouse model. The binding between LC3B and TOM20 indicates mitophagosome formation. And the binding between LC3B and Parkin suggests that Rg1 restores mitophagy through the PINK1-Parkin pathway. Besides, LIR-containing receptors including BINP3L [13], Fundc1 [12], and Bcl2L13 [14] can also directly bind to LC3B. Indeed, we investigated a relatively strong affinity between Fundc1 and LC3B in the AD cell line but not in the AD mouse model. Fundc1 and BINP3L are mainly associated with hypoxia-induced mitophagy [20,21]. The tolerance of mice to hypoxia may be higher than that of humans. This difference may alleviate Fundc1 or BINP3L-mediated mitophagy in mouse brains. We demonstrated that Rg1 might contribute to the correction of the hypoxia environment, as indicated by decreased phosphorylation levels of AMPK in Rg1-treated AD models. Therefore, few low oxygen-mediated mitophagy has been activated, and the PINK1-Parkin pathway is the major regulatory pathway in the AD mouse model.

Irreversible mitochondria dysfunction may result in cell death, which is caused by releasing cytochrome c (one of the apoptotic factors) from damaged mitochondria, followed by cell apoptosis with the high level of cleaved caspase-9 [25]. Moreover, studies have shown neuron apoptosis can be caused by APP overexpression [26]. Indeed, we observed apoptotic cells in the AD mouse hippocampus, which is merely found in Rg1-treated AD mouse and wild-type mouse hippocampus using TEM. Also, we determined the lower expression level of cleaved caspase-9 in the Rg1-treated mouse hippocampus than those in the saline-treated AD mouse hippocampus. This finding may suggest that Rg1 prevents cellular apoptosis with low expression levels of the cleaved caspase family.

Aβ deposits cause AD pathology, and a systematic review has reported that accumulation of Aβ perturbs Parkin-dependent mitophagy [27]. Although the number of Aβ deposits has no evident changes, we still identified a significant decrease in the size of the Aβ plaque. Double-staining of Aβ and Iba1 (a microglia marker) illustrated microglial phagocytosis, which leads to size reduction of Aβ deposits. This result may suggest that Rg1 diminishes the Aβ deposit by microglial phagocytosis. In turn, decreased Aβ levels may facilitate Parkin-dependent mitophagy in the Rg1-treated group.

Our studies demonstrate the neuroprotective mechanism of ginsenoside Rg1 in AD models. Rg1 induces PINK-Parkin mediated mitophagy and ameliorates memory deficits in 5XFAD mice. These findings may pave the way for the potential therapeutic effects of Rg1 for AD.

The experiments on animals were performed according to the Committee on the Use of Live Animals in Teaching and Research (CULATR, Ref. No.5196-19), The University of Hong Kong.

The work in the Li laboratory was supported by the 10.13039/501100003453Natural Science Foundation of Guangdong Province, China (2020A1515010111) and the 10.13039/501100004000Key Project of Guangzhou Science and Technology Program, China (202206010142). The work in the Song laboratory was supported by the Midstream Research Programme for Universities of Innovation and Technology Fund, Hong Kong (MRP/056/21).

Conception and design of study: N. Wang, Y.Q. Song, Z. Li; acquisition of data: N. Wang, J.Y. Yang, R.J. Chen, Y.N. Pan; analysis and/or interpretation of data: N. Wang, J.Y. Yang, Q.F. Lei, Y.Z. Wang; Drafting the manuscript: N. Wang, Y.N. Pan, S.J. Liu, L. He, Y.Y. Liu; revising the manuscript critically for important intellectual content: Y.Q. Song, Z. Li; Approval of the version of the manuscript to be published (the names of all authors must be listed): N. Wang, J.Y. Yang, R.J. C,hen, Y.Y. Liu, S.J. Liu, Y.N. Pan, Q.F. Lei, Y.Z. Wang, L. He, Y.Q. Song, Z. Li

The authors declare no competing interest.