Ginkgolide A improves the pleiotropic function and reinforces the neuroprotective effects by mesenchymal stem cell-derived exosomes in 6-OHDA-induced cell model of Parkinson’s disease

Parkinson’s disease (PD) is a common disorder attributed to the loss of midbrain dopamine (mDA) neurons and reduced dopamine secretion. Currently, the treatment regimes for PD comprise deep brain stimulations, however, it attenuates the PD progression marginally and does not improve neuronal cell death. We investigated the function of Ginkgolide A (GA) to reinforce Wharton’s Jelly-derived mesenchymal stem cells (WJMSCs) for treating the in vitro model of PD. GA enhanced the self-renewal, proliferation, and cell homing function of WJMSCs as assessed by MTT and transwell co-culture assay with a neuroblastoma cell line. GA pre-treated WJMSCs can restore 6-hydroxydopamine (6-OHDA)-induced cell death in a co-culture assay. Furthermore, exosomes isolated from GA pre-treated WJMSCs significantly rescued 6-OHDA-induced cell death as determined by MTT assay, flow cytometry, and TUNEL assay. Western blotting showed that apoptosis-related proteins were decreased following GA-WJMSCs exosomal treatment which further improved mitochondrial dysfunction. We further demonstrated that exosomes isolated from GA-WJMSCs could restore autophagy using immunofluorescence staining and immunoblotting assay. Finally, we used the alpha-synuclein recombinant protein and found that exosomes derived from GA-WJMSCs led to the reduced aggregation of alpha-synuclein compared to that in control. Our results suggested that GA could be a potential candidate for strengthening stem cell and exosome therapy for PD.


INTRODUCTION
Parkinson's disease (PD) was first characterized two hundred years ago and is one of the most common neurodegenerative disorders. Until now, it was estimated that more than 2% of the older population suffers from Parkinson's disease [1]. This percentage is expected to rise with the aging of the population worldwide and a few reports state that PD would become a pandemic [2]. The pathological features of PD include the loss of midbrain dopamine (mDA) neurons or neuronal cell loss in the substantia nigra (SN), decreased dopamine secretion, and Lewy body accumulation in other brain tissues, thereby exacerbating motor functions [3][4][5][6][7]. The pathophysiology of PD remains unknown but may involve genetic and environmental risks. The most effective treatments for PD involve deep brain stimulations through surgery or taking some enzyme inhibitors and levodopa [8]. However, long-term drug usage may reduce its efficacy and lead to side effects involving involuntary motor action and dyskinesia, which affects patients' quality of life.
Studies on stem cell therapy in PD have been popular for the past decades. Mesenchymal stem cells (MSCs) are the most appropriate for stem cell therapy because of two main characteristics: the immuno-modulatory function and the differentiation ability [9]. MSCs can be isolated from bone marrow, adipose tissues, umbilical cord Wharton's Jelly (WJ), and even the dental pulp, which are classified as "mesenchymal resources" and have inspired researchers to find their abilities in cell transplantation application for PD. The secretome of stem cells has been found to play an important role in several degenerative diseases [10]. Increasing evidence has revealed that extracellular vesicles (EVs) play multiple roles in various physiological conditions, especially through cell-cell communication. EVs from MSCs include microvesicles, apoptotic bodies, and exosomes. The exosomes consist of genetic substances (DNA, RNA, and non-coding RNA), proteins, enzymes, and some growth factors [11]. Exosomes from MSCs can regulate different cell signaling pathways in target cells and promote their biological activities by transmitting information and content to improve disease conditions [12].
Here, we aimed to understand the role of GA in Wharton's Jelly MSCs to aid in restoring the 6hydroxydopamine (6-OHDA)-induced neuronal cell death. Hence, we assessed the self-renewal, homing, and exosome-mediated functions of MSCs treated with GA, which may be a potential compound to be used in stem cell or secretome therapy.

Cell culture
Wharton's Jelly MSCs were obtained from Cellular Engineering Technologies Inc. (CET, Coralville, IA, USA) and SH-SY5Y cells obtained from the American Type Culture Collection (Manassas, VA, USA) were cultured in Dulbecco's modified essential media (Life Technologies) with 10% fetal bovine serum (FBS, Hyclone Thermo Fisher Scientific) and 1% penicillinstreptomycin (Gibco). Cells were incubated at 37°C with 5% CO2 in a humidified incubator.

MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay
Cells were seeded in 24-well plates (1-3 × 10 4 cells/well) and cultured in a complete DMEM medium for 12 h. Further, they were treated with 6-OHDA for 6-48 h at 37°C. Cells were washed twice with phosphate-buffered saline (PBS) and incubated with the MTT reagent (Invitrogen ™ ) for 2 h. At the end of the treatment, cells were incubated in Dimethyl sulfoxide and shaken for 10-15 min. Cell viability was analyzed autophagy using immunofluorescence staining and immunoblotting assay. Finally, we used the alpha-synuclein recombinant protein and found that exosomes derived from GA-WJMSCs led to the reduced aggregation of alpha-synuclein compared to that in control. Our results suggested that GA could be a potential candidate for strengthening stem cell and exosome therapy for PD.
as the optical density (OD) was determined spectrophotometrically at a wavelength of 570 nm.

Transwell co-culture assay
To assess the effect of WJMSCs on neuroblast cells, in vitro co-culture assay was performed where WJMSCs (2 × 10 4 cells) were placed in the upper chamber (0.4 μm), and SH-SY5Y (3 × 10 4 cells) cells were seeded in the lower well (24 well plate). SH-SY5Y cells were treated with 6-OHDA and incubated at 37°C for 24-48 h. The SH-SY5Y cell viability were to be determined.

Transwell homing assay
To examine the in vitro homing ability, WJMSCs (1 × 10 4 cells) were seeded in the upper chamber of transwell migration chambers and SH-SY5Y (3 × 10 4 ) cells in the lower well containing 500 µL of medium with 6-OHDA and incubated at 37°C for 24 h. Later, cells were removed by a cotton swab from the transwell membrane and cells on the surface of the well were fixed using 4% paraformaldehyde (PFA). The fixed cells were washed in PBS and stained with crystal violet. Cells were counted and the migration ability was expressed as fold-change compared to control.

Flow cytometry assay for marker expression and apoptosis/cell cycle assessment
SH-SY5Y or WJMSCs were washed twice with PBS then incubated with 5 μL FITC Annexin V and propidium iodide reagent for 15 min at RT in the dark according to the instruction. The apoptotic cells were analyzed using flow cytometry where 10,000 events were acquired in BD FACSLyric ™ system. For marker expression, WJMSCs with or without GA treatment were collected and fixed with 4% paraformaldehyde solution then washed twice with PBS. WJMSCs were then stained with Vimentin (Santa Cruz; sc-32322, 1:100) for 30 min and incubated with secondary FITCconjugated goat anti-mouse IgG (Invitrogen; A11001, 1:100) in the dark for 30 min. The cells were acquired in BD FACSLyric ™ system.

Exosome isolation assay
Exosomes were collected from the culture medium of WJMSCs using an Exosome Purification Kit (Exospin ™ ) as per the manufacturer's protocol. The culture medium was centrifuged at 300 × g for 10 min to remove cells and debris. The supernatant was then transferred to a fresh centrifuge tube and spun at 20,000 × g for 30 min to remove any cell debris. The exosomes were then precipitated at 4°C for 24 h. The exosome-containing pellet (resuspended in 200 µL PBS) was added to the column and centrifuged at 50 × g for 60 s.

Immunofluorescence staining
Briefly, alpha-synuclein (5 µM, active, Pre-Formed Fibrils, GeneTex) were treated to the SH-SY5Y cell along or not along with 6-OHDA for 24 hours. The cells were then fixed in 4% PFA, washed twice with PBS, and immunostained with a specific primary antibody (1:50). The cells were then incubated with a fluorescence conjugated secondary antibody (1:50) then used the DAPI mounting gel (Abcam, ab104139) to cover the slides and visualized with a fluorescence microscope (BX40, Olympus) as previously described [19]. AGING

Statistical analyses
All experiments were repeated at least three times and statistical analysis was evaluated using SigmaPlot 12.5 software (Systat Software, Inc., San Jose, CA, USA). Student t-test or one-way analysis of variance (ANOVA) was used to analyze the differences in the data. P < 0.05 was considered as significant.

Data availability statement
The data generated from this manuscript are available from the corresponding author on reasonable request.

GA promotes proliferation and stemness of WJMSCs
The potential effect of GA on WJMSC proliferation was investigated by treating with different concentrations of GA. After 5 d of treatment, GA (80 µM) promoted WJMSCs proliferation ( Figure 1A) as examined by MTT assay. We further assessed the changes in cell cycle phases after GA treatment and found that it could change the cell cycle distribution in S and G2/M phases as compared to that in controls ( Figure 1B). In addition, the expression of proliferation markers, p-AKT, PCNA, Cyclin E, and Cyclin B1, was increased after GA treatment ( Figure 1C). GA also promoted the stemness markers, OCT4 and Nanog, in WJMSCs ( Figure 1D) and maintained the MSC marker Vimentin expression ( Figure 1E). These findings indicated that GA acts in improving the self-renewal ability of the stem cells.

GA enhances the homing ability of WJMSCs
In order to explore the multiple functions of GA in stem cells, we further examined the homing ability in GA pre-treated WJMSC (24 h) by co-culturing with SH-SY5Y cells in a 6-OHDA damage model. WJMSCs pretreated with 80 µM GA was seeded into the inserts (2 × 10 5 cells/insert) of a transwell. SH-SY5Y cells were pre-plated into the lower wells with 100 µM of 6-OHDA. The migrating WJMSCs counted after 24 h revealed that GA pre-treated WJMSCs displayed higher migrating capacity compared to that of the untreated controls ( Figure 2A). Moreover, GA enhanced CXCR4 expression instead of CXCR7 in WJMSCs ( Figure 2B). These results suggested that GA could promote WJMSCs homing ability through the SDF-1/CXCR4 axis which is in the line with previous studies that showed preconditioned MSCs required CXCR4 for cell migration whereas CXCR7 for survival in damage area [20,21]. GA pre-treated WJMSCs effectively protect SH-SY5Y cells from 6-OHDA induced apoptosis 6-OHDA is a well-known chemical that induces the death of dopaminergic neurons by oxidative stress. To determine whether GA can promote the WJMSCs to rescue SH-SY5Y cells in the 6-OHDA damage model, we performed transwell co-culture assay as previously described [22]. 6-OHDA decreased SH-SY5Y cell viability and the concentration of 100 µM was chosen for further studies ( Figure 3A). Further, GA pretreatment in WJMSCs significantly mitigated the death of SH-SY5Y cells under 6-OHDA stimulation ( Figure  3B). These findings suggested that GA pre-treated WJMSCs might repair the damage through secretomes, cytokines, or exosomes. To further clarify the mode of action, we utilized GW4869, an inhibitor of exosome generation which blocked the exosome production, in the co-culture assay. Here, a reduction in the GA pretreated WJMSCs-dependent protection was observed after GW4869 treatment ( Figure 3C). These data suggest that exosomes play an important role in GAmediated WJMSCs therapeutic function.

Effects of GA pre-treated WJMSC-derived exosomes on the 6-OHDA damage model
To gain insight into the WJMSC exosome function enhanced by GA, exosomes were isolated from WJMSCs with or without 80 µM GA pre-treatment, and their diameters were examined by the NanoSight (NS300, Malvern Panalytical) ( Figure 4A). Further, the presence of the exosome markers tumor susceptibility gene 101 (TSG101) and CD81 and the negative marker calnexin was confirmed ( Figure 4B). We found that the exosomes derived from GA pre-treated WJMSCs enhanced SH-SY5Y cell viability under 6-OHDA treatment than that in control WJMSCs using MTT assay ( Figure 4C). Flow cytometry and TUNEL assay also demonstrated that exosomes derived from GA pretreated WJMSCs markedly reduced SH-SY5Y cell apoptosis ratio compared to that of control ( Figure 4D-4F). Tyrosine hydroxylase (TH) also revealed that THpositive cells were decreased under 6-OHDA treatment but could be preserved by exosomes derived from GA pre-treated WJMSCs ( Figure 4G).

GA pre-treated WJMSC-derived exosomes suppress apoptosis markers and impair mitochondrial homeostasis
The above results illustrated that GA displays multiple functions and repairs the 6-OHDA damage cell model. We further investigated the mechanism to support the functional assays. Exosomes isolated from GA pretreated WJMSCs could decrease apoptosis-related AGING   The data is represented as the mean values ± standard deviation. Significance ascribed as * P < 0.05, ** P < 0.01 vs. control. AGING protein Caspase-9 and cleaved Caspase-3 expression but increased anti-apoptosis protein Bcl-2 expression ( Figure 5A). Moreover, it also showed that exosomes isolated from GA pre-treated WJMSCs can reduce cytochrome C and Bax expression under 6-OHDA stimulation ( Figure 5B). Therefore, we examined the OXPHOS markers and identified that exosomes isolated from GA pre-treated WJMSCs can restore the mitochondrial homeostasis observed during the 6-OHDA damage in SH-SY5Y cells ( Figure 5B, lower panel).
GA pre-treated WJMSC-derived exosomes recover excessive autophagy in the 6-OHDA model and promote alpha-synuclein clearance 6-OHDA treatment in neuronal cells leads to abnormal autophagic flux and causes cell apoptosis [23]. Hence, we investigated the autophagic flux in the cell damage model. The LC3 dots were abundant under 6-OHDA treatment. However, the puncta in SH-SY5Y cells can be recovered similar to that of control after treatment with the GA pre-treated WJMSC derived exosomes ( Figure 6A). Immunoblotting assay showed higher Beclin-1 and LC3B but reduced p62 protein expression under 6-OHDA stimulation. Treatment with the exosomes from GA pre-treated WJMSCs restored the autophagic dysregulation similar to that observed in the control group (without 6-OHDA) ( Figure 6B). To further validate our findings, we treated the SH-SY5Y cells with alpha-synuclein (5 µM, active, Pre-Formed Fibrils, GeneTex) under 6-OHDA stimulation which led to increased alpha-synuclein aggregation in the 6-OHDA group. However, GA pre-treated WJMSCsderived exosome treatment significantly reduced the alpha-synuclein aggregation, suggesting that it could promote alpha-synuclein clearance through improved autophagic flux ( Figure 6C).

DISCUSSION
Most of the Ginkgolide derivatives from Ginkgo biloba are known for their properties such as anti-inflammatory, neuroprotective, and anti-cancer effects [24][25][26]. GA, GB, and GC could protect against cerebral ischemia/reperfusion damage in rats through Nrf2/CREB regulation [27]. GB promoted neuronal differentiation through the Wnt/β-catenin pathway in neural stem cells from mice [28]. It was hence applied in several platforms for enhancing cell differentiation and Parkinson's model [29][30][31][32]. Although various functions of GA have been determined, such as antiatherosclerosis by preventing oxidative stress [33]; and anti-neurotoxic by preventing Tau hyperphosphorylation [17], direct evidence showing GA function in stem cells is lacking. Our study is the first to contribute to revealing that GA has potential benefits like GB in stem cells but in promoting stemness rather than cell differentiation. Therefore, GA can provide a new strategy to maintain stem cell properties for clinical application. In this study, we demonstrated a novel finding to use the natural compound GA that promoted the proliferation, self-renewal, and homing ability of WJMSCs, and further enhanced the exosomes secreted from WJMSCs to repair 6-OHDA-induced dopaminergic neuronal cell death. GA pre-treated WJMSCderived exosomes could not only inhibit 6-OHDA induced excessive autophagy by regulating autophagy-related protein thus recover mitochondrial complex I/III/V but balancing the autophagy functions in alpha-synuclein clearance and protecting the cell from apoptosis (Figure 7).
Stem cell therapy is popular due to its self-renewal ability and differentiation into various cell lineages. It brings hope for the treatment of diseases which cannot be cured by drug therapy. However, there are some disadvantages that need to be overcome such as the allogenic graft-dependent rejection. The paracrine effect of the cytokines or the secretome and immunomodulation is more important than the MSCs themselves. Exosomes derived from different types of stem cells have been utilized in the treatment of numerous disease models [34]. Using compounds/ components with a specific function in regenerative medicine gave rise to its unique worth, which could improve its outcome. The study showed that exosomes from Fisetin-treated keratinocytes could promote hair growth in an animal model [35]. Adiponectin stimulated exosome production by hMSCs to protect against heart failure in mice [36]. Here, we found that GA can enhance the function of exosomes from WJMSCs to mitigate cell death in a Parkinson's disease cell model, providing a better outcome than those isolated from untreated WJMSCs.
The Parkinson's disease in vitro models that include MPP+ and 6-OHDA treatment in neuroblastoma cells are well established with known mechanisms. Exosomes isolated from WJMSCs have been shown to repair cell damage caused by 6-OHDA-induced autophagic dysfunction [22]. Several studies also highlighted that 6-OHDA led to excessive autophagy that resulted in the release of cytochrome C from the mitochondria [23]. In our study, we found the exosome derived from GA pre-treated WJMSCs can return excessive autophagy to the normal state, thereby restoring OXPHOS ( Figures 5C, 6A and 6B). 6-OHDA treatment in SH-SY5Y induced LC3/Beclin 1 but decreased p62 expression which confirmed autophagy activation in our model. However, exosome derived from GA pre-treated WJMSCs reversed the autophagyrelated protein expression implying that the exosomes could inhibit autophagy thus protect neuronal survival. Interestingly, GA pre-treated WJMSCs derived exosomes brought about a significant clearance of the alpha-synuclein protein in 6-OHDA treatment ( Figure  6C). These data reveal that the protective effects of exosomes might be attributed to their efficiency in stabilizing autophagy even under 6-OHDA stimulation. Therefore, analyzing the contents of the exosomes is key to ascertain the potential components (such as proteins, mRNAs, or non-coding RNAs from the donor MSCs) and mechanisms that mediate their beneficial effects. The average size of GA pre-treated WJMSCs exosome was larger than that of the control MSCs ( Figure 4A). It might hint to the presence of more "cargo," which is promoted by GA treatment in the WJMSCs to secrete or aid in exosome biogenesis. The specific and potential targets in GA pre-treated WJMSCs exosome with a high repair capacity will be examined in future experiments.

CONCLUSION
We found a potential compound, GA, that can not only promote WJMSCs stemness and homing ability but also enhance exosome function to repair Parkinson's disease in vitro. The beneficial effects were mediated through GA's restoration of autophagy function to clear the alpha-synuclein protein and repair mitochondrial dysfunction (Figure 7). This study aids in providing evidence for implementing stem cell-derived exosomal therapy in the PD treatment regime.

CONFLICTS OF INTEREST
The authors declare no conflicts of interest related to this study.