Plant sphingolipids promote extracellular vesicle release and alleviate amyloid-β pathologies in a mouse model of Alzheimer’s disease

www.nature.com/scientificreports OPEN Plant sphingolipids promote extracellular vesicle release and alleviate amyloid-β pathologies in a mouse model of Alzheimer’s disease Kohei Yuyama1*, Kaori Takahashi2, Seigo Usuki1, Daisuke Mikami1, Hui Sun1, Hisatoshi Hanamatsu3, Junichi Furukawa3, Katsuyuki Mukai2 & Yasuyuki Igarashi1 The accumulation of amyloid-β protein (Aβ) in brain is linked to the early pathogenesis of Alzheimer’s disease (AD). We previously reported that neuron-derived exosomes promote Aβ clearance in the brains of amyloid precursor protein transgenic mice and that exosome production is modulated by ceramide metabolism. Here, we demonstrate that plant ceramides derived from Amorphophallus konjac, as well as animal-derived ceramides, enhanced production of extracellular vesicles (EVs) in neuronal cultures. Oral administration of plant glucosylceramide (GlcCer) to APP overexpressing mice markedly reduced Aβ levels and plaque burdens and improved cognition in a Y-maze learning task. Moreover, there were substantial increases in the neuronal marker NCAM-1, L1CAM, and Aβ in EVs isolated from serum and brain tissues of the GlcCer-treated AD model mice. Our data showing that plant ceramides prevent Aβ accumulation by promoting EVs-dependent Aβ clearance in vitro and in vivo provide evidence for a protective role of plant ceramides in AD. Plant ceramides might thus be used as functional food materials to ameliorate AD pathology. Alzheimer’s disease (AD) is a common form of dementia with a pathology characterised by the progressive intracerebral accumulation of amyloid-β protein (Aβ). This accumulation results from impaired clearance of Aβ in the sporadic form of AD and from increased production due to genetic mutations of amyloid precursor protein (APP) or Aβ processing enzymes in the less-common familial form of AD1. The imbalanced metabolism of Aβ that results in its accumulation is linked to tau pathology, neuronal impairment and the eventual emergence of the clinical symptoms of AD2. The prolonged preclinical stages of AD, including the phase of Aβ accumulation, provide a critical opportunity for preventive interventions. Small extracellular vesicles (40–100 nm in diameter) called exosomes are released from various types of cells that have recently emerged as a key player for the intercellular transport of molecules both in health and in disease conditions, including AD3,4. In neuronal cultures and sera from AD patients, EVs associate with amyloid precursor protein (APP) and its metabolites, including C-terminal fragments (CTFs), amyloid intracellular domain and Aβ5–7. Our previous studies showed that extracellular vesicles (EVs) released from cultured neurons associate with Aβ through their surface glycosphingolipids (GSLs) and are incorporated into microglia for degradation8,9. Furthermore, APP transgenic mice infused continuously with neuron-derived EVs had decreased Aβ levels and amyloid deposition in their brains, suggesting that EVs mediate Aβ clearance9,10. We also reported that production of EVs in neurons is modulated by the metabolism of the sphingolipid family of membrane lipids. The inhibition of neutral sphingomyelinase-2 reduces ceramide and prevents exosome release, whereas knockdown of sphingomyelin synthase 2 (SMS2) increases ceramide and promotes exosome release8,11. 1 Lipid Biofunction Section, Faculty of Advanced Life Science, Hokkaido University, Kita-21, Nishi-11, Kita-ku, Sapporo, 001-0021, Japan. 2R & D Headquarters, Daicel Corporation, 2-18-1, Konan, Minato-ku, Tokyo, 108-8230, Japan. 3Department of Advanced Clinical Glycobiology, Faculty of Medicine, Graduate School of Medicine, Hokkaido University, Kita-21, Nishi-11, Kita-ku, Sapporo, 001-0021, Japan. *email: Scientific Reports | (2019) 9:16827 | https://doi.org/10.1038/s41598-019-53394-w 1 www.nature.com/scientificreports/ www.nature.com/scientificreports Figure 1. Plant ceramides induced release of neuron-derived EVs. (a) Chemical structures of typical GlcCer extracted from Amorphophallus konjac and its deviated ceramide. (b) Particle numbers of the EVs isolated from supernatants of SH-SY5Y cells treated for 24 h with GlcCer or ceramides. (c) Particle size distribution of the control and GlcCer- and ceramide-treated fractions of EVs. (d) Western blotting for Alix, CD63 and ganglioside GM1 exosome markers in supernatants from SH-SY5Y cells (1 × 105/lane) and isolated EVs (from 1 × 107 cells/ lane). (e) Exosome amounts in culture supernatants of GlcCer- or ceramide-treated SH-SY5Y cells measured by PS-capture exosome ELISA system. (f) Particle numbers of the EVs isolated from supernatants of SH-SY5Y cells or primary neurons treated with the indicated concentrations of ceramides for 24 h. Data are presented as means ± SDs. **P < 0.01; ***P < 0.001 by t tests. One of the major sphingolipids that is digested daily by humans is plant glucosylceramide (GlcCer)12. GlcCer, a type of GSL, has a single glucose attached to ceramide, which is hydrolysed into its components, glucose, a fatty acid and a sphingoid base, by digestive intestinal enzymes for uptake by intestinal enterocytes13. A portion of the sphingoid bases, including those of plant origins, is then resynthesized to ceramide, GlcCer and other complex sphingolipids such as sphingomyelin14,15. Sphingoid bases have diverse structures16. The most common sphingoid base in mammalian cells is sphingosine (trans-4-sphingenine, d18:14), whereas plant sphingolipids consist of Δ8-unsaturated sphingoid bases such as 4,8-sphingadienine (d18:24,8)17,18. Recent reports indicate that dietary plant sphingolipids have beneficial effects, providing improvements to the skin barrier and anticancer activity19–22. In this study, we examined the effect of plant ceramides on EVs-dependent Aβ clearance to prevent AD pathogenesis. We showed that oral administration of plant GlcCer to APP transgenic mice markedly reduced Aβ levels and amyloid plaques and eventually attenuated Aβ-related pathologies, such as inflammation, synaptic dysfunction and cognitive deficits. We also demonstrated that plant sphingolipids increase the production of neuron-derived EVs with the ability to clear Aβ in neuronal cultures and in mice. Results Exogenously added plant ceramides increased release of EVs from neuronal cultures. To examine the effects of plant ceramides on production of EVs, we treated human neuroblastoma SH-SY5Y cells with Amorphophallus konjac-derived GlcCer and ceramides at a concentration of 10 µM for 24 h. The ceramides were prepared from the GlcCer by glucose hydrolysis with EGCases (Fig. 1a)18. We previously showed that the sphingoid bases of konjac-derived GlcCer and Cer from these are composed of a major 4,8-sphingadienine (d18:24,8) and, an minor 4-hydroxy-8-sphingenine (t18:18), with combinations of C16:0, C18:0 and C20:0 fatty acids (Fig. 1a)18. Scientific Reports | (2019) 9:16827 | https://doi.org/10.1038/s41598-019-53394-w 2 www.natu (...truncated)