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 . 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 Aβ1−42 (JKM2, hAβ1−42) 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 hAβ1−42 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 hAβ1−42 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 . 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, both hAβ1−42 and hTau[P301L] models exhibited a shorter lifespan in comparison to WT control (Fig. 1b). Upon PEP administration, only hAβ1−42 nematodes displayed a significant extension of lifespan, with no influence of PEP extract on the lifespan of hTau[P301L] and the WT animals (Fig. 1c-e). A summary of the lifespan data in different groups was in Supplementary Table 1. These findings indicate PEP protected against memory deficits and extended lifespan in particular the hAβ1−42 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 hAβ1−42 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 [32–34]. Cholinergic neurons play a key role in the CNS, and acetylcholine (ACh) works as neurotransmitter that serviced all cholinergic neurons. There is a likelihood that either Ach depletion or hyper-accumulation links to neurodegeneration [35–37]. 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 . 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 hAβ1−42 model of AD displayed an increased sensitivity to aldicarb compared to the WT N2; whilst 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 hAβ1−42 nematodes. Application of PEP resulted in a delay in aldicarb-mediated paralysis in both hAβ1−42 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 sub-type neurons, we used a series of well-characterized nematode models whereby hAβ1−42 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)] ewere used for data quantification as these five neurons show clear, stable and easy-to-quantify patterns of neurodegeneration . As reported before [27, 42], transgenic nematodes carrying hAβ1−42 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).
Encouraged by the strong in vivo glutamatergic neuronal protection by PEP, we asked whether this benefit is preserved in mammalian cells including in the HT-22 and SH-SY5Y cells. To this end, mammalian HT-22 mouse hiPEPocampal cells (undiffentiated), which are devoid of cholinergic and glutamate receptors, were utilized to evaluate glutamate-induced cell death and examine the neuroprotective effect of PEP via checking cell viability using the MTT assay. Glutamate at 5 mM showed a significant toxicity (around 45%) in the HT-22 cells compared to vehicle control (Fig. 2g-j). Exposing the HT-22 cells to PEP extract as co-treatment with glutamate resulted in a dose-depended inhibition of HT-22 cell death (Fig. 2g). Furthermore, 6h- and 12h-pretreatment with PEP showed even better cell protection against glutamate toxicity (5 mM, 24 h) (Fig. 2h-i). In addition to use HT-22 cells, we further studies neuroprotective effects of PEP using the human fibroblastoma SH-SY5Y cells. We used the two-step retinoic acid (RA) and brain-derived neurotrophic factor (BDNF) protocol and successfully differentiate the SH-SY5Y cells to neuronal-like cells (Supp. Fig. 1b-d), followed by Aβ toxicity assay. Aβ25−35 peptides reduced cell viability in dose-dependent manners compared to vehicle control (Supp. Fig. 1e). Co-administration of PEP at 100 µg/ml (but not lower doses as we tested), with Aβ25−35 or as pre-treatment at 6 hours (but not 12 hours) prior to Aβ25−35 administration, was sufficient to protect against Aβ25−35-induced neuronal death (Fig. 3a-c). Our data suggest PEP protects against Aβ25−35-induced cellular death in both mouse and human neuronal-like cells.
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 sub-type 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-SY5Y-differentiated neuronal-like cells. Immunoblot data showed that PEP (100 µg/mL, 6h pre-treatment) inhibited phosphorylation of the mammalian target of rapamycin (mTOR) (Fig. 3d, e), reduced phosphorylation of ULK1 at p-S757 (activation of this site inhibits ULK1 activity), and increased the expression of the lysosome protein Cathepsin D; compared with the Aβ25−35 group, Aβ25−35 + PEP did not have significant effects on the protein levels of PINK1, Parkin, p62, SOD-1 or SOD2 (Fig. 3d-i).
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 mitochondria-targeted GFP together with the autophagosomal marker LC3/LGG-1 fused with DsRed [10, 47]. Normally, mitophagy-inducing stimuli encourage the formation of autophagosomes that extensively co-localize with mitochondria . Here, we demonstrate a pronounced induction of mitophagy via formation of autophagosomes consisting of mitochondria upon PEP exposure (Fig. 4a-b). This implies that PEP was able to promote the formation of mito-autophagsosmes 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 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 . 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 . PEP was indeed able to stimulate mitophagy as the mtRosella animals displayed significantly decreased GFP/DsRed ratio compared to vehicle controls (Fig. 4c-d). Combined the human cell data and the nematode data, we propose PEP stimulates mitophagy via activating the key mitophagy/autophagy protein ULK1 and increasing the expression of lysosome protein Cathepsin D.
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 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 (UPRmt)’, and ‘oxidative stress’, which are linked to neuroprotection [49, 50]. Surprisingly, 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 hAβ1−42 model of AD and in the WT N2 (Fig. 5a-b). This could be due to technical limitation as the whole nematode tissue was used for RNA extraction while the cells we were interested in, the total neurons (302 neurons), only constitutes around 10% of the total cells in a hermaphrodite nematode. However, significant upregulation of genes associated with oxidative stress (gst-4 and sod-3) as well as the mitochondrial unfolded protein response (UPRmt) (ubl-5) were observed in the N2 animals (Fig. 5a). Whilst the hAβ1−42 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 . Previous studies reported that DAF-16 (ortholog for the mammalian FOXO transcription factors) is the upstream regulator of gst-4 and sod-3 [52, 53]. In our nematode system, the expression level of daf-16 gene was not changed in either the N2 controls or the hAβ1−42 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 solely in 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 and e). 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 protects against Aβ-induced memory loss is daf-16 dependent
To further 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. N2neu−sid1 and hAβ1−42(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 N2neu−sid1 animals, but also abolished memory restoration ability of PEP extract in both N2neu−sid1 and hAβ1−42(JKM2) neu−sid1 animals. However, sod-3 RNAi only abolished memory restoration of PEP in hAβ1−42(JKM2) neu−sid1 animals but had no effect in healthy control N2neu−sid1 animals (Fig. 5f, g). Collectively, PEP inhibited memory deficits through upregulation of the DAF16-SOD3 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 was shown in supplementary Table 2. To further narrow down the 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) [54, 55]. 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, 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 that 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), tocopherols including α-tocopherol (-8.556 kcal/mol), ϒ-tocopherol (-8.356 kcal/mol), and δ-tocopherol (-8.227 kcal/mol) showed the highest binding potential to the insulin like protein in C. elegans. Other compounds such as stigmast-4-en-3-one (-8.246 kcal/mol), squalene (-8.186 kcal/mol) and cholest-4-en-3-one (-8.151 kcal/mol), α-amyrin (-7.874 kcal/mol), as well as β-amyrin (-7.453 kcal/mol) could form a stable complex with 2KJI; these data suggest that attenuation of the insulin pathway via these compounds may activate DAF-16 nuclear translocation. Further wet laboratory experiments are necessary to identify the compound(s) that could induce mitophagy and forestall memory loss and attenuate pathologies in AD animals.