Evaluation of Plant Ceramide Species-Induced Exosome Release from Neuronal Cells and Exosome Loading Using Deuterium Chemistry

The extracellular accumulation of aggregated amyloid-β (Aβ) in the brain leads to the early pathology of Alzheimer’s disease (AD). The administration of exogenous plant-type ceramides into AD model mice can promote the release of neuronal exosomes, a subtype of extracellular vesicles, that can mediate Aβ clearance. In vitro studies showed that the length of fatty acids in mammalian-type ceramides is crucial for promoting neuronal exosome release. Therefore, investigating the structures of plant ceramides is important for evaluating the potential in releasing exosomes to remove Aβ. In this study, we assessed plant ceramide species with D-erythro-(4E,8Z)-sphingadienine and D-erythro-(8Z)-phytosphingenine as sphingoid bases that differ from mammalian-type species. Some plant ceramides were more effective than mammalian ceramides at stimulating exosome release. In addition, using deuterium chemistry-based lipidomics, most exogenous plant ceramides were confirmed to be derived from exosomes. These results suggest that the ceramide-dependent upregulation of exosome release may promote the release of exogenous ceramides from cells, and plant ceramides with long-chain fatty acids can effectively release neuronal exosomes and prevent AD pathology.


Introduction
Alzheimer's disease (AD) is the most common neurocognitive disorder and is a progressive neurodegenerative disorder that affects more than 50 million people worldwide. Senile plaques of aggregated amyloid-β (Aβ), produced from amyloid precursor protein (APP) following consecutive cleavage reactions by βand γ-secretases, accumulate in brains, and this is the initial pathological stage of AD [1,2]. Therefore, drugs that inhibit Aβ processing, such as inhibitors of βand γ-secretases, have been developed. However, despite several decades of drug discovery research and clinical trials, progress in AD treatments has advanced slowly. Additionally, since Aβ deposition begins long before the onset of cognitive deficits, precisely diagnosing early AD pathology is difficult. It is reported that functional lipid ingestion in this preclinical term may be an effective strategy for AD prevention. For instance, epidemiological research has linked the consumption of high omega-3 fatty acids such as docosahexaenoic acid (DHA) and eicosatetraenoic acid (EPA) with a lower risk of AD [3]. Our recent study demonstrated that the oral administration of glucosylceramides (GlcCer), extracted from the konjac root, into AD model mice attenuated the Aβ burden in the brain and improved cognitive activities [4].
(EPA) with a lower risk of AD [3]. Our recent study demonstrated that the oral ad istration of glucosylceramides (GlcCer), extracted from the konjac root, into AD m mice attenuated the Aβ burden in the brain and improved cognitive activities [4]. A tionally, the oral intake of plant GlcCer for six months lowered blood biomarkers alleviated brain amyloid burden in human subjects with healthy and mild cognitive pairment (MCI) [5].
Exosomes, a type of small extracellular vesicles, are derived from the endos membrane. Our previous reports showed that exosomes released from cultured neu possess the potential for mediating Aβ clearance [4,[6][7][8][9]. Neuronal exosomes asso with Aβ through their surface glycosphingolipids and are incorporated into microgli degradation. In addition, we reported that exosome release from SH-SY5Y cells is moted by treatment with exogenous mammalian-type ceramides (Cers), and this invo the lysosome-associated protein transmembrane 4B (LAPTM4B) [10]. Importantly length of the acyl chain influences activity; Cers consisting of long acyl chains (C16:0 C18:0) enhance exosome release, whereas those with shorter and longer chains ha effects. Therefore, the structural characteristics of Cer species may be a crucial fact promoting exosome release. However, it remains to be explored how different plan species induce exosome production. In plant tissues (rice, corn, and konjac root), sphingoid bases exist as D-erythro-(4E,8Z or 8E)-sphingadienine (d18:2) or D-erythrophytosphingenine (t18:1), while most mammalian Cers ( Figure 1) exist as D-erythro-sp gosine (d18:1). Focusing on the structures of plant Cers could reveal crucial aspects explain their pharmacological activities. In the present work, we found that plant including D-erythro-(4E,8Z or 8E)-sphingadienine, accelerated exosome release mor fectively than mammalian-type Cers that were linked to d18:1. Additionally, struc differences in the fatty acyl chains of plant Cers affected the exosome's release; the acyl chains most strongly stimulated exosome release, whereas geometric isomeris the C8-C9 position had no effect. Moreover, tracking using liquid chromatography spectrometry (LC-MS) with penta-deuterium-labeled plant Cers revealed that exoge Cers were derived from exosomes.

Identification of Plant Cer Types Promoting Exosome Release
One of the major classes of sphingolipids in the human diet is the plant GlcCer [11]. In this study, we selected eight major species of plant GlcCer isolated from konjac (Amorphophallus konjac) and purified them by enzymatic glucose cleavage according to a previously reported method [12]. As shown in Figure 2, no changes in cell viability were observed in SH-SY5Y cells after the treatment with each of the eight Cer species, up to 50 µM.

Identification of Plant Cer Types Promoting Exosome Release
One of the major classes of sphingolipids in the human diet is the plant GlcCer [11]. In this study, we selected eight major species of plant GlcCer isolated from konjac (Amorphophallus konjac) and purified them by enzymatic glucose cleavage according to a previously reported method [12]. As shown in Figure 2, no changes in cell viability were observed in SH-SY5Y cells after the treatment with each of the eight Cer species, up to 50 μM.
We previously demonstrated that neuronal exosome-bound Aβ is taken up by microglia, transported through the endocytic pathway, and degraded in lysosomes [6]. To determine whether the increase in EVs induced by Cers d18:2/18 h:0 treatment promotes Aβ clearance, a transwell culture system, in which exosomes and Aβ secreted from Aβoverexpressing SH-SY5Y cells placed on upper inserts can interact with microglial BV-2 cells placed at the bottom of the wells, was utilized. Under this experimental setting, we added plant or mammalian Cer species to the cultures at ten µM, and after 24 h of co-incubation, the levels of Aβ in the medium were determined. The levels of both Aβ40 and Aβ42 in the culture media significantly decreased following Cer d18:2/18 h:0 and Cer d18:1/18:0 treatment, but not Cer d18:2/20 h:0 and Cer d18:2/24 h:0 ( Figure 4). In addition, the concentrations of Aβ40, a major species of Aβ, were much lower after the treatment with Cer d18:2/18 h:0 than Cer d18:1/18:0 treatment, suggesting that plant Cers have high potency relative to exosome-dependent clearance extracellular Aβ. Exosomes were measured using a PS-Capture Exosome ELISA system. Results were normalized against controls and represented as the mean ± SD (n = 3; * p < 0.05, ** p < 0.01, *** p < 0.001 by one-way ANOVA).
We previously demonstrated that neuronal exosome-bound Aβ is taken up by microglia, transported through the endocytic pathway, and degraded in lysosomes [6]. To determine whether the increase in EVs induced by Cers d18:2/18 h:0 treatment promotes Aβ clearance, a transwell culture system, in which exosomes and Aβ secreted from Aβoverexpressing SH-SY5Y cells placed on upper inserts can interact with microglial BV-2 cells placed at the bottom of the wells, was utilized. Under this experimental setting, we added plant or mammalian Cer species to the cultures at ten μM, and after 24 h of coincubation, the levels of Aβ in the medium were determined. The levels of both Aβ40 and Aβ42 in the culture media significantly decreased following Cer d18:2/18 h:0 and Cer d18:1/18:0 treatment, but not Cer d18:2/20 h:0 and Cer d18:2/24 h:0 ( Figure 4). In addition, the concentrations of Aβ40, a major species of Aβ, were much lower after the treatment with Cer d18:2/18 h:0 than Cer d18:1/18:0 treatment, suggesting that plant Cers have high potency relative to exosome-dependent clearance extracellular Aβ.  Exogenous mammalian-type Cers promote exosome release through LAPTM4B, transmembrane proteins expressed in late endosomes and lysosomes [10]. Previous studies reported that synthetic d18:1-type Cers bind to recombinant LAPTM4B protein. To determine whether LAPTM4B is also involved in plant Cers-dependent exosome release, we performed a siRNA-mediated knockdown study. LAPTM4B knockdown in SH-SY5Y cells completely prevented the increase in exosome release induced by d18:2/18 h:0, as well as d18:1/18:0 (Figure 5a). A protein-ceramide overlay assay using an NH2-terminal glutathione S-transferase (GST) fusion LAPTM4B protein revealed that apparent binding Exogenous mammalian-type Cers promote exosome release through LAPTM4B, transmembrane proteins expressed in late endosomes and lysosomes [10]. Previous studies reported that synthetic d18:1-type Cers bind to recombinant LAPTM4B protein. To determine whether LAPTM4B is also involved in plant Cers-dependent exosome release, we performed a siRNA-mediated knockdown study. LAPTM4B knockdown in SH-SY5Y cells completely prevented the increase in exosome release induced by d18:2/18 h:0, as well as d18:1/18:0 (Figure 5a). A protein-ceramide overlay assay using an NH 2 -terminal glutathione S-transferase (GST) fusion LAPTM4B protein revealed that apparent binding signals were obtained for d18:2 4E,8Z/18 h:0 (Figure 5b). The signal densities were higher than those for d18:1/18:0, suggesting an intense association between plant Cers and LAPTM4B (Figure 5c).

Discussion
In this study, we investigated the abilities of plant Cer species to stimulate exosome release from SH-SY5Y cells. The ability of plant Cers to induce exosome release varied with structural differences. Regarding fatty acid components, Cer species with long-chain fatty acids (C16 and C18) strongly promoted exosome release, whereas those with very long-chain fatty acids (C20, C22, and C24) did not exert an effect. Regarding sphingoid base components, plant-type moieties stimulated exosome release more effectively than mammalian-type components (d18:1/18:0 vs. d18:2/18 h:0, Figure 3a). According to the correlation between exosome release and the Cer-LAPTM4B interaction [10,15,16], the length of the fatty acid is the most significant element for Cer to bind its interaction motif Results were represented as the mean ± SD (n = 3; ** p < 0.01).

Discussion
In this study, we investigated the abilities of plant Cer species to stimulate exosome release from SH-SY5Y cells. The ability of plant Cers to induce exosome release varied with structural differences. Regarding fatty acid components, Cer species with long-chain fatty acids (C16 and C18) strongly promoted exosome release, whereas those with very long-chain fatty acids (C20, C22, and C24) did not exert an effect. Regarding sphingoid base components, plant-type moieties stimulated exosome release more effectively than mammalian-type components (d18:1/18:0 vs. d18:2/18 h:0, Figure 3a). According to the correlation between exosome release and the Cer-LAPTM4B interaction [10,15,16], the length of the fatty acid is the most significant element for Cer to bind its interaction motif in LAPTM4B; additionally, the plant sphingoid base may possess a higher affinity for LAPTM4B than mammalian sphingosine and/or assist protein-protein interactions between LAPTM4B and other downstream signaling agents, thereby inducing exosome production. Therefore, LAPTM4B could be considered to recognize the molecular structure of Cer for its binding. The most common sphingoid bases in mammals and plants have two chiral centers at the C-2 and C-3 positions. Theoretically, there are four stereoisomers, D-erythro, L-erythro, D-threo, and L-threo, in the sphingoid base, but only the D-erythro type has been found. In fact, L-threo type ceramides were reported to exhibit a higher affinity towards a lipid metabolism enzyme compared to the D-erythro type [17]. Thus, focusing on the stereochemistry of sphingoid bases becomes one of the fascinating studies for developing higher potential materials to promote exosome release through LAPTM4B.
In the present study, we also prepared deuterated plant Cer-d 5 (4E,8Z d18:2-d 5 /18:0) to investigate its localization in SH-SY5Y cells, and LC-MS results revealed that exogenously added plant Cers were~3-fold enriched in exosomes compared with cells at 24 h after treatment. We previously demonstrated that Cer species are directed to endosomal compartments, such as multivesicular bodies, and transported for recycling and exosome release through LAPTM4B [10]. In addition, LAPTM4B is primarily localized to late endosomes and lysosomes [18,19]. Therefore, this result indicates that vesicle trafficking and exosomal efflux may be triggered by the interaction of Cers with LAPTM4B localized mainly in late endosomes.
Finally, the intake of plant sphingolipids from daily food and natural sources can lead to skin barrier improvement, such as decreased transepidermal water loss and enhanced stratum corneum flexibility [20]. GlcCer is hydrolyzed into its components (glucose, a fatty acid, and a sphingoid base) by intestinal digestive enzymes for uptake by intestinal enterocytes [21]. Some sphingoid bases, including those of plant origin, are then resynthesized to Cers, GlcCers, and other complex sphingolipids, such as sphingomyelins, and then absorbed into the body [22][23][24]. Our previous study demonstrated that synthetic plant-type Cers could permeate into brains through the blood-brain barrier (BBB) in mouse and BBB cell culture models [25]. Therefore, the oral intake of plant sphingolipids and sphingoid bases as dietary supplements could help prevent AD by enhancing neuronal exosome release. There are various sphingoid bases in nature, such as sphinganine, phytosphingosine, 9-methyl sphingadienine, and sphingatrienine. The results of the present study indicate that the diverse activities of Cers reflect structural differences, and this knowledge may pave the way for developing Cer species that are more effective for AD prevention or therapy. Such sphingoid bases could serve as new sphingoid-based chemotherapeutics.  22 (m, 42H), and 0.87 (t, J = 6.9 Hz, 3H). 13

Cell Culture and Treatments
Neuroblastoma SH-SY5Y cells were maintained at 37 • C with 5% CO 2 in Eagle's minimum essential medium/Ham's F-12 medium (ThermoFisher Scientific, Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS). Primary neuronal cultures were prepared from cerebral cortices of embryonic day 15 mice using a dissociation solution (Sumitomo Bakelite, Tokyo, Japan). The cells were plated on polyethyleneimine-coated dishes and cultured for 7 days in neurobasal medium with 25 mM KCl, 2 mM glutamine, and B27 supplement (ThermoFisher Scientific, Waltham, MA, USA). ReproNeuro, a neuron progenitor derived from human iPSCs, was purchased from ReproCELL (Yokohama, Japan) and maintained in ReproNeuro maturation medium for 14 days according to the manufacturer's instructions.

Exosome Collection and Quantification
Exosomes were collected from culture supernatants of SH-SY5Y cells by centrifuging the culture at 2000 g for 10 min and then centrifuging at 10,000 g for 30 min at 4 • C to remove cells and debris. The supernatant was then centrifuged at 100,000 g for 1 h at 4 • C to obtain exosomes as pellets. Exosomes were measured by PS-Capture Exosome ELISA (Fujifilm Wako, Osaka, Japan) according to the manufacturer's instructions. Antibodies against human CD63 and mouse CD9 (MAB5218, R&D Systems, Minneapolis, MN, USA) were used for SH-SY5Y cell and human iPS neuron-derived EVs and primary mouse neuron-derived EVs, respectively. The size and number of exosomes were measured using a qNano nanoparticle analyzer (Izon Science, Cambridge, MA, USA). CPC100 served as a calibration sample.

Ceramide Overlay Assay
Recombinant human LAPTM4B containing a GST-tag at the N-terminus (Abnova, Taipei, Taiwan) was used in this study. In brief, 10 pmol of ceramides was spotted on nitrocellulose membranes. After the blocking with Blocking One (Nacalai tesque, Kyoto, Japan), the membranes were incubated with 1 nM LAPTM4B overnight at 4 • C. LAPTM4B bound to the ceramides on the membrane was detected using an HRP-conjugated anti-GST antibody. The dot densities were quantified using ImageJ software 2.0.0 (Bethesda, MD, USA).