P. edulis Extract Protects Against Amyloid-β Toxicity in Alzheimer’s Disease Models Through Maintenance of Mitochondrial Homeostasis via the FOXO3/DAF-16 Pathway

Alzheimer’s disease (AD) is a common and devastating disease characterized by pathological aggregations of beta-amyloid (Aβ) plaques extracellularly, and Tau tangles intracellularly. While our understandings of the aetiologies of AD have greatly expanded over the decades, there is no drug available to stop disease progression. Here, we demonstrate the potential of Passiflora edulis (P. edulis) pericarp extract in protecting against Aβ-mediated neurotoxicity in mammalian cells and Caenorhabditis elegans (C. elegans) models of AD. We show P. edulis pericarp protects against memory deficit and neuronal loss, and promotes longevity in the Aβ model of AD via stimulation of mitophagy, a selective cellular clearance of damaged and dysfunctional mitochondria. P. edulis pericarp also restores memory and increases neuronal resilience in a C. elegans Tau model of AD. While defective mitophagy-induced accumulation of damaged mitochondria contributes to AD progression, P. edulis pericarp improves mitochondrial quality and homeostasis through BNIP3/DCT1-dependent mitophagy and SOD-3-dependent mitochondrial resilience, both via increased nuclear translocation of the upstream transcriptional regulator FOXO3/DAF-16. Further studies to identify active molecules in P. edulis pericarp that could maintain neuronal mitochondrial homeostasis may enable the development of potential drug candidates for AD.


Introduction
Alzheimer's disease (AD) is a progressive and irreversible disease of the central nervous system (CNS). It is the most common form of dementia affecting around 50 million people globally at present, a figure estimated to triple by 2050 [1]. Clinically, it is characterized by an insidious onset and progressive deterioration of cognitive function [1,2]. The pathological hallmarks of the disease include formation of extracellular plaques composed of aggregated beta-amyloid (Aβ) and accumulation of intracellular Tau in the form of neurofibrillary tangles [3][4][5][6][7]. These pathological features are accompanied by neuroinflammation, mitochondrial dysfunction, synaptic degeneration, and neuronal loss due to necroptosis [8][9][10][11][12][13]. To date, cholinesterase inhibitors and glutamate receptor antagonists have been the standard drugs for the treatment of AD. These therapeutic interventions provide symptomatic relief, but are incapable of curing and/ or delaying the progression of the disease. Therefore, there is a dire need for identification of novel therapeutic strategies to counter AD.
Passiflora edulis (P. edulis), commonly known as passion fruit, is native to Southern America, but widely cultivated in tropical and subtropical areas worldwide. The pulp and pericarp of the passion fruit are a source of phytochemical contents such as polyphenols, triterpenoids, glycosides, carotenoids, polysaccharides, aromatic oils, and essential nutrients [14][15][16][17][18]. Pharmacological studies have identified the bioactivities of passion fruit including anti-oxidative, anti-inflammatory, anti-diabetic, and potentially hepatoprotective effects [19][20][21][22][23]. Additionally, it has been reported that passion fruit extracts act as a modulator of the glutamatergic system, which further promotes neuroprotective activities [24,25]. However, the underlying mechanism of the neurotherapeutic activity of P. edulis extract has remained elusive. In this study, we wanted to determine whether administration of P. edulis extract could inhibit memory loss and pathological phenotypes in Caenorhabditis elegans (C. elegans) models of AD. We further evaluated the underlying molecular mechanisms in both C. elegans and mammalian cell systems.

P. edulis Pericarp Extract Attenuates Memory Loss and Prolongs Lifespan in AD C. elegans
Progressive memory impairment is the most common symptom in AD patients [26]. Thus, we set out to evaluate whether the P. edulis pericarp (PEP) extract can inhibit memory loss in the transgenic C. elegans models of AD harboring pan-neuronal human Aβ 1-42 (JKM2, hAβ  or pan-neuronal expression of human P301L Tau mutation (CK12, hTau[P301L]). For this purpose, we utilized an aversive olfactory learning chemotaxis assay (a negative value correlates with positive chemotaxis-related memory). Transgenic nematodes expressing hAβ  and hTau[P301L] displayed severe cognitive deficits and neurodegeneration as we [10,27] and others [28,29] reported before. We administrated PEP at 250 µg/ml to the nematodes from egg hatching onwards and performed memory experiments on adult day 1. While the hAβ  and hTau[P301L] animals had impaired memory, PEP inhibited memory loss in these AD nematodes; to note, PEP did not influence the memory of WT (N2) animals (Fig. 1a). Epidemiological studies indicate that AD not only impairs memory but also shortens lifespan [30,31]. We postulated that strategies that improve memory in animals with AD could also extend their lifespan [27]. Therefore, we subsequently assess the potential effect of PEP on lifespan in the transgenic nematode models of AD. As expected, in the transgenic C. elegans models of AD, especially, the hTau[P301L] model exhibited a shorter lifespan in comparison to WT control (Fig. 1b). Upon PEP administration, not only hTau[P301L] and hAβ  nematodes displayed a significant extension of lifespan, but also elongate the lifespan of the WT animals ( Fig. 1c-e). Along with lifespan, we also evaluated pharyngeal pumping in adult day 2 and day 8 animals. PEP supplementary with no influence on pumping rate (Fig. 1f, h) indicated that PEP extended lifespan not due to the starvation. A summary of the lifespan data in different groups is shown in Supplementary Table 1. These findings indicate PEP protected against memory deficits and extended lifespan in particular the hAβ 1-42 model of AD.

P. edulis Extract Inhibits Neurodegeneration in AD C. elegans and Cells
Having established the potential of PEP extract to improve healthspan and lifespan in the C. elegans hAβ 1-42 model of AD, we set out to investigate the underlying mechanism. For this purpose, we first evaluated whether PEP potentiates neuroprotection that results in the improved functional behavior. The two major neurotransmission systems primarily affected in AD are the cholinergic and the glutamatergic systems [32][33][34]. Cholinergic neurons play a key role in the CNS, and acetylcholine (ACh) works as a neurotransmitter that serviced all cholinergic neurons. There is a likelihood that either Ach depletion or hyper-accumulation links to neurodegeneration [35][36][37]. The functional activity of the cholinergic system in the AD nematodes was assessed by feeding the animals with aldicarb, an acetylcholinesterase inhibitor that induces hyper-accumulation of Ach, resulting in accelerated skeletal muscle contraction and finally paralysis [38]. Controls for the assay, in the form of aldicarb hypersensitive (VC233: tom-1(ok285)I) and aldicarb-resistant (NM204: snt-1(md290)II) strains, displayed increased and reduced sensitivities to aldicarb, respectively, compared to the WT nematodes (Fig. 2a). The hAβ 1-42 model of AD displayed an increased sensitivity to aldicarb compared to the WT N2; while hTau[P301L] nematodes did not show increased sensitivity to aldicarb compared to that of WT animals (Fig. 2a). These findings suggest an impairment in the cholinergic system in hAβ  nematodes. Application of PEP resulted in a delay in aldicarb-mediated paralysis in both hAβ  and hTau[P301L] models of AD, as well as in the WT N2 nematodes (Fig. 2b-d). This implies that PEP enhanced cholinergic neuronal resistance to aldicarb in both pathological and physiological conditions.
In addition to cholinergic neuronal protection, we asked whether PEP could protect against degeneration of the glutamatergic neurons in AD. Glutamatergic neurons are another vital type of neurons found in the CNS, and are impaired in AD [39,40]. Aβ induces glutamatergic neuronal loss and promotes AD progresses [40,41]. To evaluate whether PEP could protect against Aβ-induced neurodegeneration in the glutamatergic subtype neurons, we used a series of well-characterized nematode models whereby hAβ  is only expressed in the glutamatergic neurons and induces neurodegeneration [27,42]. Five tail-localized glutamatergic neurons (LUA(R), LUA(L), PVR, PLM(R), and PLM(L)) were used for data quantification as these five neurons show clear, stable, and easy-to-quantify patterns of neurodegeneration   [42]. As reported before [27,42], transgenic nematodes carrying hAβ 1-42 overexpression in their glutamatergic neurons exhibited significant reduction in glutamatergic neurons in comparison to controls, implicating Aβ-mediated neurodegeneration of glutamatergic neurons in the models of AD (Fig. 2e, f). PEP administration almost completely annulled Aβ-induced neurodegeneration (Fig. 2e, f).

P. edulis Extract Increased Neuronal Mitophagy in Human Neurons and C. elegans
Compromised mitophagy-induced accumulation of damaged mitochondria in the brain, especially in the entorhinal cortex and the hippocampus, is an early sign and a risk factor of AD [8,10,27]. Our previous studies show that genetic or pharmacological restoring of neuronal mitophagy abrogated memory loss and pathologies in AD [10,27]. Here, we asked whether PEP could induce mitophagy, and if yes, whether PEP-induced memory retention is dependent on mitophagy activation. Mitophagy is a subtype of selective autophagy; thus, there are many proteins participating in both cellular events [43,44]. For mechanistic exploration, we checked expression levels of proteins critical for the mitophagy and autophagy pathways using the SH-SY5Ydifferentiated neuronal-like cells. Immunoblot data showed that PEP (100 μg/ml, 6-h pretreatment) inhibited phosphorylation of the mammalian target of rapamycin (mTOR) (Fig. 4a, b), reduced phosphorylation of ULK1 at p-S757 (activation of this site inhibits ULK1 activity), and increased the expression of mitophagy-related multifunctional protein BNIP3 and the lysosome protein cathepsin D; compared with the Aβ 25-35 group, Aβ 25-35 + PEP did not have significant effects on the protein levels of PINK1, Parkin, p62, SOD-1, or SOD-2 ( Fig. 4a, b).
While the immunoblot data strongly suggest a possibility that PEP affects mitophagy/autophagy proteins, we further designed experiments to validate this possibility. To investigate that PEP could induce mitophagy in neurons, we utilized two composite systems for monitoring mitophagy in vivo [45,46]. First, we utilized transgenic animals expressing a mitochondriatargeted GFP together with the autophagosomal marker LC3/ LGG-1 fused with DsRed [10,46]. Normally, mitophagyinducing stimuli encourage the formation of autophagosomes that extensively co-localize with mitochondria [46]. Here, we demonstrate a pronounced induction of mitophagy via formation of autophagosomes consisting of mitochondria upon PEP exposure (Fig. 4c, d). This implies that PEP was able to promote the formation of mito-autophagosomes for mitochondria cargo for degradation via mitophagy. Next, we wanted to establish whether the mitochondria in the autophagosome were indeed degraded. For this purpose, we , and PVR were used for data analysis. Data were from three biological repeats. One-way ANOVA followed by Tukey's multiple comparisons test was used for data analysis with ns, no significance; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. g-i PEP attenuated high glutamate (5 mM)-induced cell death in HT-22 cells under different conditions. Varied concentrations (12.5-100 µg/ ml) of PEP were used in the experiments. Data were from three biological repeats. One-way ANOVA followed by Tukey's multiple comparisons test was used for data analysis with ns, no significance; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.
◂ utilized transgenic animals expressing mitochondria-targeted Rosella (mtRosella) biosensor that combines a fast-maturing pH-insensitive DsRed fused to a pH-sensitive green fluorescent protein (GFP) variant [47]. Mechanistically, quenching of the GFP signal upon uptake of the mitochondrial cargo by the acidic lysosome is indicated by a lower GFP/DsRed ratio representing mitophagy stimulation [46]. PEP was indeed able to stimulate mitophagy as the mtRosella animals displayed significantly decreased GFP/DsRed ratio compared to vehicle controls (Fig. 4e, f). Combining the human cell data and the nematode data, we propose PEP stimulates mitophagy via activating the key mitophagy/autophagy protein ULK1, and

P. edulis Extract Promotes Mitochondrial Homeostasis and Oxidative Resistance via DAF-16 Nuclear Translocation in C. elegans
In addition to the mechanism mentioned above, we wondered whether PEP-based neuronal benefits could be started at the transcriptional level. We used real-time PCR and checked the mRNA levels of a list of genes in the groups of "mitophagy," "mitochondrial unfolded protein response (UPR mt )," and "oxidative stress," which are linked to neuroprotection [48,49]. PEP did not induce significant change, despite an upward trend in genes associated with mitophagy (i.e., pdr-1, dct-1, lgg-1, and skn-1) in the hAβ 1-42 model of AD and in the WT N2 (Fig. 5a, b). However, significant upregulation of genes associated with oxidative stress (gst-4 and sod-3) as well as the mitochondrial unfolded protein response (UPR mt ) (ubl-5) was observed in the N2 animals (Fig. 5a). While the hAβ 1-42 model of AD exhibited upregulation of only sod-3 upon PEP application (Fig. 5b), SOD-3 has a role in suppressing oxidative stress that underlies mitochondrial and cellular dysfunction [50]. Previous studies reported that DAF-16 (orthologue for the mammalian FOXO transcription factors) is the upstream regulator of gst-4 and sod-3 [51,52]. In our nematode system, the expression level of the daf-16 gene was not changed in either the N2 controls or the hAβ 1-42 model of AD (Fig. 5a, b). Therefore, we went on to investigate whether PEP supplementation could promote the nuclear translocation of DAF-16 by using a transgenic nematode with a DAF-16::GFP-tag. A 4-point grading system was utilized for characterizing DAF-16 localization from the cytosol (1) to predominant nuclear localization (4) (Fig. 5c, d). Under physiological conditions, DAF-16 was distributed predominantly in the cytoplasm; however, upon stimulation with heat-shock (a positive control), DAF-16 was mainly localized in the nucleus (Fig. 5c). PEP induced significant nuclear translocation of DAF-16 (Fig. 5c, d). Altogether, our data suggest that PEP upregulates gst-4 and sod-3 genes via enhancing subcellular distribution of DAF-16 from the cytoplasm to the nucleus, resulting in increased DAF-16-regulated transcription activity.

P. edulis Extract Induces Neuronal Mitophagy and Protects Against Aβ-Induced Memory Loss Which is daf-16 Dependent
To further investigate whether PEP induces mitophagy in a daf-16/dct-1/sod-3-dependent manner or not, we knocked down daf-16, dct-1, and sod-3, respectively, via RNAi feeding of the animals from the egg hatching stage. The mtRosella neu−sid1 (to knockdown target only in the neurons but not other tissues) transgenic animals were used in these experiments. Our results show that knock down of daf-16 abolished PEP-induced neuronal mitophagy in mtRosella neu−sid1 animals; similar results were shown in dct-1 or sod-3 knocked down animals (Fig. 5e, f). In addition, to investigate whether the neuroprotective effect of PEP is daf-16 dependent or not, we knocked down the daf-16 or sod-3 gene by using RNAi feeding of the animals from egg hatching. N2 neu−sid1 and hAβ 1-42 (JKM2) neu−sid1 transgenic animals were used in these experiments, and our results suggested that knock down of daf-16 gene expression not only caused memory deficits in healthy control N2 neu−sid1 animals, but also abolished memory restoration ability of PEP extract in both N2 neu−sid1 and hAβ 1-42 (JKM2) neu−sid1 animals. However, sod-3 RNAi only abolished memory restoration of PEP in hAβ 1-42 (JKM2) neu−sid1 animals but had no effect in healthy control N2 neu−sid1 animals (Fig. 5g,  h). Cumulatively, PEP protects neurons in AD animals via upregulation of the DAF-16/DCT-1/SOD3-dependent mitophagy pathway.

Identification of Potential Bioactive Compounds in P. edulis Pericarp
The neuroprotective effect of PEP could be contributed by the small bioactive compounds inside the extracts. To identify small molecules in PEP, we used gas chromatography-mass spectrometry (GC-MS). Over hundreds of compounds have been identified in PEP extract and the list of compounds is shown in Supplementary Table 2. To further narrow down the Data were from at least three biological repeats. One-way ANOVA followed by Tukey's multiple comparisons test was used for data analysis with ns, no significance; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. d Representative images showed the oxidized MitoSOX fluorescence signal in control, Antimycin A1 (Anti-A1), Aβ [25][26][27][28][29][30][31][32][33][34][35] , and PEP pre-or co-treated with Aβ [25][26][27][28][29][30][31][32][33][34][35] in differentiated SHSY5Y cells. Scale bar, 20 µm. e PEP alleviated Aβ 25-35 -induced mitochondrial superoxide level. Anti-A1 (100 μM) was used as a positive control. Data were from three biological repeats. One-way ANOVA followed by Tukey's multiple comparisons test was used for data analysis with ns, no significance; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. f PEP pretreatment (6 h ahead) attenuated Aβ 25-35 -reduced mitochondrial membrane potential. FCCP (20 Μm, 1 h) was used as positive control. Data were from three biological repeats. One-way ANOVA followed by Dunnett's multiple comparisons test was used for data analysis with ns, no significance; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.  LGG-1::DsRed DCT-1::GFP L C3 II/I list of potential candidates which might inhibit AD pathologies and own translational potential, we considered capacity of compound candidates to pass the blood-brain barrier (BBB) [53,54]. We used the SwissADME software to predict BBB permeability of all compounds. As a result, 15 compounds were highly ranked with BBB permeability (Table 1). While the compound phenol showed the highest BBB permeability score in this system, others such as squalene, tocopherols, and amyrins may have high affinity to BBB receptor(s) in the BBB permeant system. Since PEP extract enhanced DAF-16 nuclear localization, a computer docking analysis was used to predict whether PEP extract containing potential compounds could induce FOXO3/DAF-16 nuclear translocation in different conditions, including via inhibiting the insulin/ IGF-1 signaling pathway. 2KJI, an insulin-like protein found in C. elegans, was used as a target protein, and the docking analysis was performed on the top 10 potential compounds in the list. For the results, a higher negative binding energy indicates a higher stability of the protein-ligand complex. In our study (Supplementary Table 3

Discussion and Conclusion
Where there is no drug available to cure AD, turning up mitophagy is suggested as a promising strategy for anti-AD drug development [10,27,55]. Here, we demonstrate that PEP extract inhibits Aβ 25-35 -induced mitochondrial superoxide production and loss of MMP, which then attenuated neuronal cell death. PEP extract not only increases neuronal mitophagy level and alleviates neurodegeneration, but also inhibits memory impairment in AD C. elegans, especially in the hAβ 1-42 model of AD. In particular, we show these benefits to be mediated by the nuclear localization of DAF-16, which stimulates mitophagy and protects against oxidative stress. FOXO3/ DAF-16 is a fundamental component of the insulin/IGF signaling (IIS) pathway, which plays a critical role in longevity and stress resistance in various organisms including in humans [56][57][58][59]. In C. elegans, DAF-16 not only regulates longevity and dauer development, but is also involved in metabolism and stress resistance. The activity of DAF-16 is regulated by the upstream protein, DAF-2 (orthologue of the mammalian insulin and insulin-like growth factor-1 receptor) [60,61]. Upon activation, DAF-16 disconnects from the 14-3-3 proteins that negatively regulates the insulin-like signaling (IIS) pathway and is positively regulated by the JNK pathway [62]. Upon translocation to the nucleus, DAF-16 promotes target gene expression of transmembrane tyrosine kinase (old-1) [63], glutathione-S-transferase 4 (gst4) [64,65], BNIP3/NIX/dct-1 [47,66], and sod-3 [67,68]. Here, we show that the DAF-16-regulated downstream genes, including gst-4 and sod-3, were increased upon PEP supplementation. Previous studies suggested that activated neuronal DAF-16 elicits intestinal DAF-16 activation, and vice versa [69,70]. Here, our results showed that PEP increased DAF-16 nuclear translocation. In turn, activated DAF-16 directly promotes sod-3 expression level. To note, although enhanced DAF-16 activity, we did not detect significant change of dct-1; this could be caused by the use of whole worm tissue for the PCR rather than to use the isolated neurons. Related experiments could be performed using a neuronal isolation protocol for tissue collection in the future. Interestingly, our immunoblot data show that PEP activated ULK1, an essential protein involved in both autophagy and mitophagy [43,71], via inhibiting mTOR. Additionally, PEP increased the expression levels of BNIP3 (the mammalian homolog of the C. elegans dct-1) and the lysosome protein cathepsin D; upregulation of these mitophagy/lysosome proteins could enhance mitophagy. In line with this, two in vivo mitophagy quantification assays unambiguously support the possibility of a neuronal mitophagy induction capacity by PEP via the DAF-16/DCT-1/SOD3 pathway. Importantly, knocked Quantifications of the expression level of designated proteins as compared to GAPDH. Data were from three biological repeats. One-way ANOVA followed by Tukey's multiple comparisons test was used for data analysis with ns, no significance; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. c Representative images showing the LGG-1 and DCT-1 co-localization in control or PEP (250 µg/mL) extract fed adult day 1 nematodes. Scale bar, 10 µm. d PEP enhanced LGG-1 and DCT-1 co-localization which indicate mitophagy events. Data were from two biological repeats with a total of 38 to 45 nematodes used for data quantification. One-way ANOVA followed by Tukey's multiple comparisons test was used for data analysis with ns, no significance; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. e Representative images showing the GFP/DsRed ratio in control or PEP (250 µg/ml) extract fed adult day 1 mtRosella nematodes. Scale bar, 50 µm. f PEP reduced GFP/DsRed ratio indicating increased mitophagy. Data were from three biological repeats with 40 nematodes. One-way ANOVA followed by Tukey's multiple comparisons test was used for data analysis with ns, no significance; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Original western blot gels for a are included in Supplementary Fig. 2  Taking all the pieces of data together, it suggests that PEP could regulate both mitophagy and mitochondrial resilience, which may contribute to memory retention and neuroprotection in the AD animals via DAF-16 dependent pathways (Fig. 6).
Until here, the neuroprotective effects and underlying mechanism of PEP extract have been partially uncovered in this study. By extrapolation, it is likely that multiple small compounds in PEP that contributed to the beneficial effects. Fortunately, our findings clearly show P. edulis pericarp could be a good source of bioactive compounds, and potential compounds of PEP extract were identified in this study. In the future, it will be interesting to continue studying the therapeutic ability of the potential candidates in multiple AD models.

Plant Collection and Preparation
The fresh passion fruit (P. edulis 'Paul Ecke') was collected from Chiang Mai, Thailand, and identified by the herbarium of Kasin Suvatabhandju (Department of Botany, Faculty of Science, Chulalongkorn University, Thailand) with the voucher specimen [016437 B(CU)]. Pericarp was cut and air-dried before being ground into a fine powder. P. edulis pericarp powder was macerated in petroleum ether with a ratio of 1:10. The PEP extract was filtered through Whatman No. 1 filter paper and concentrated using vacuum distillation and a rotary evaporator. Stock solution was prepared in DMSO (concentration is 100 mg/ml).

Cell Differentiation
SH-SY5Y cell differentiation was induced using a previously described protocol with slight modifications [72]. Briefly, at day 0, SHSY5Y cells were seeded into the experimental plates with normal culture medium and cultured overnight. On day 1, we replace the normal culture medium by using DMEM/F12 medium (5% FBS) with retinoic acid (RA) (10 µM) to initiate cell differentiation, and change to DMEM/F12 medium (2.5% FBS) with RA (10 µM) to stimulate further cell differentiation in day 2 to day 5. Additionally, DMEM/F12 medium (0% FBS) with 50 ng/ mL brain-derived neurotrophic factor (BDNF) was used to strengthening cell differentiation in day 6 to day 8. Then, the differentiated cells are ready for experiments on day 9.

Cell Viability
3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) is a widely used chemical indicator for cellular metabolic activity and cell viability based on the ability of nicotinamide adenine dinucleotide phosphate (NADPH)-dependent cellular oxidoreductase enzymes which reduce the yellow MTT to purple formazan in living cells. The formazan redissolved in the solubilization solution such as DMSO and provides a colorimetric assay. In this study, MTT was used to detect cell viability for both HT-22 and SHSY5Y cells. For HT-22 cells, 5000 cells were seeded and grown in each well of 96-well plates overnight in a humidified 5% CO 2 incubator at 37 °C. Next day, the cells were pretreated (6 h, 12 h) or co-treated with varied concentrations of PEP extract (12.5 to 100 µg/ mL) and glutamate (5 mM). At the end of the drug treatment time, cells were exposed to MTT for 3 h. Then, all supernatant was removed, and the formazan crystals were dissolved with DMSO (200 µL) before measuring the absorbance at 550 nm. For SHSY5Y cells, the differentiated cells were used for cell viability detection. As mentioned earlier, cells are ready to use after a 9-day differentiation period, and pretreated (6 h, 12 h) or co-treated with varied concentrations of PEP extract (12.5 to 100 µg/mL) and Aß [25][26][27][28][29][30][31][32][33][34][35] (25 µM). At the end of the drug treatment time, cells were exposed to MTT for 3 h. Then, the synthesized formazan crystals were dissolved with DMSO (200 µL) before measuring the absorbance at 550 nm.

Gas Chromatography-Mass Spectrometry (GC-MS) and Theoretical Prediction of Blood-Brain Barrier (BBB) Permeability of Compounds Analysis
The beneficial effects of the PEP extract are due to the various potential compounds. GC-MS was employed to

C. elegans Strains
C. elegans strains were maintained on Escherichia coli OP50 using the standard feeding methods. The temperature for  Table 3.

Drug Treatment of C. elegans
The PEP extract or equal volume of DMSO was directly added in the melted NGM before being poured into the plates. Nematodes were treated from either egg hatching or L4 stage.

Toxicity Assay
A toxicity assay was used to determine the safe dose of PEP extract selection in C. elegans. N2, a wild-type strain, was used for these experiments. At day 1, ten of 1-day-old nematodes were placed on the NGM plates with OP50 which contained PEP extract (0.025, 0.25, 0.5 mg/mL) or not (vehicle) for 3-h egg laying. The number of eggs was counted after adults were removed. On day 2, the number of L1 larvae and unhatched eggs was counted to check the egg hatching efficiency. In day 3, the number of L4 larvae was counted. On day 4, the number of 1-day-old adult nematodes was counted. Each group includes three technical repeats.

Lifespan and Healthy Span Analysis
AD caused health issues and reduction of lifespan has been reported [1,73]. We recorded the living time to check whether PEP extract have beneficial effects for lifespan extension in healthy (N2) nematodes and AD human Tau[P301L] and human Aβ 1-42 transgenic nematodes. The animals were synchronized by bleaching and grown at 20 °C until the L4 stage. Twenty of L4 stage animals were picked and placed in each experiment plate (with or without PEP extract). FUDR was added to prevent egg hatching and animals were transferred to the fresh plates for every 2-3 days until day 10, and then every 5-6 days (if food running out) until death. The number of living or dead animals was recorded every day until the last animal's death. Every experiment included 3 technical repeats. Additionally, C. elegans drawing food through its pharynx and the times of pharyngeal contraction and relaxation indicate the food uptake rate. Along with the lifespan experiment, the pharyngeal pumping rate of day 2 and day 8 nematodes was evaluated via manually counting for 30 s, and 10 animals were randomly selected from each technical repeat.

Chemotaxis Behavior Assay
Isoamyl alcohol (IA), a volatile liquid, was employed to perform the chemotaxis assay as previously described [74,75]. Briefly, animals were synchronized by bleaching and grown on the OP50 seeded NGM plates with or without PEP extract at 20 °C until day 1. Animals (200 to 300 nematodes/ group) were collected and washed 4 times with MilliQ water, and then placed on conditional NGM plates (no OP50) with/ without IA on the middle of lid (10 µL) for 90 min. After that, animals were washed and transferred to the start point in the experimental plates and the number of animals from each area was recorded after 2 h. The experimental plate (10 cm) is separated into three main areas, which were labeled as IA, T (trap point), and S (start point). A small piece of Parafilm was placed in the middle of the "IA" area and 3 µl of 2% IA was topped on the Parafilm. The chemotaxis index was calculated as ("IA" -"T") / ("IA" + "T" + "S").

Aldicarb Assay
Aldicarb, an acetylcholinesterase inhibitor, was employed to evaluate the sensitivity of C. elegans to the synaptic transmission of acetylcholine at the neuromuscular junction. The C. elegans strains VC233 and NM204 grown on the OP50 seeded NGM plates were used as hypersensitive and resistant control in the experiment, respectively. Animals for experiments were synchronized by bleaching and grown on the OP50 seeded NGM plates with or without PEP extract at 20 °C until day 1. Thirty animals were transferred on each NGM plate with 0.75 mM aldicarb, and the non-paralyzed animals were recorded every 30 min for the aldicarb-induced paralysis. Each experimental group includes 3 biological repeats and 3 technical repeats.

Glutamatergic Neurons Imaging
Glutamatergic neurodegeneration was detected in C. elegans using the previous reported method. Animals were synchronized by bleaching and grown on the OP50 seeded NGM plates with or without PEP extract at 20 °C until adult day 3. There are about 15 glutamatergic neurons in the worm tail region, and 5 were selected in this study, which are LUA(R), LUA(L), PVR, PLM(R), and PLM(L). The tail regions of day 3 animals were imaged using a confocal microscope. Each experimental group includes 2 biological repeats and 3 technical repeats.

Screening of Neuronal Mitophagy in C. elegans
Two C. elegans strains were employed to quantify mitophagy induction potential of PEP extract in C. elegans. For both experiments, the nematodes were prepared and placed on the OP50 seeded NGM plates with or without PEP extract (250 µg/ml) from the egg hatching stage. The first transgenic animal expressing LGG-1::DsRed (autophagosomal marker), together with DCT-1::GFP (mitophagy reporter) in neurons. The double positive animals (adult day 1) were paralyzed by levamisole, mounted on 4% agarose pads, and imaged using confocal microscopy. The co-localization of LGG-1 and DCT-1 was count for mitophagy events. Another transgenic animal expressing pan-neuronal mitophagy reporter (mt-Rosella biosensor) represents the mitophagy level according to the ratio between pH-sensitive GFP to pH-insensitive DsRed (the lower the ratio, the higher the mitophagy events). Day 1 animals were paralyzed by levamisole, mounted on 4% agarose pads, and imaged using a confocal microscope at 10 × or 40 × magnification. Each experimental group includes 2 to 3 biological repeats and 3 technical repeats.

DAF-16 Nuclear Localization
Animals were synchronized by bleaching and grown on the OP50 seeded NGM plates at 20 °C until L4 stage. Animals were transferred to the OP50 seeded NGM plates with or without PEP extract for 24 h. The DAF-16::GFP nuclear translocation of the adult day 1 nematodes was paralyzed by levamisole, mounted on 4% agarose pads, and imaged using a confocal microscope at 40 × magnification. Each experimental group includes 2 biological repeats and 2 technical repeats. The nuclear localization level was scored as level 1 to level 4.

RNA Interference (RNAi) by Feeding
Feeding bacteria expressing dsRNA (feeding RNAi) was used to knock down selected targets (daf-16 and sod-3) in C. elegans. Briefly, selected bacteria were grown in the LB (50 mg/mL ampicillin) and added on the NGM plates containing 1% ampicillin and 1% IPTG with/without PEP extract. The animals were synchronized by bleaching or egg laying and grown on these RNAi plates until adult day 1 for chemotaxis behavior experiments and neuronal mitophagy screening assay, respectively.

Statistical Analysis
All results presented in this study have at least two biological repeats, except lifespan (one biological repeat with three technical repeats). For the imaging base experiments, data were quantified using ImageJ software. And statistical data was analyzed using Prism 8.0 software. The data were presented in mean ± SEM. The difference between the two treatment groups was analyzed using unpaired t-tests. And the group differences were analyzed using one-way ANOVA with Tukey's multiple comparisons test. The difference for multiple targets was analysis using two-way ANOVA with Sidak's multiple comparisons test. P < 0.05 is considered as statistically significant.