Ceramide levels controlled by the sphingomyelin (SM) cycle have essential roles in cancer cell fate through the regulation of cell proliferation, death, metastasis, and drug resistance. Recent studies suggest that exosomes confer cancer malignancy. However, the relationship between ceramide metabolism and exosome-mediated cancer malignancy is unclear. In this study, we elucidated the role of ceramide metabolism via the SM cycle in exosomes and drug resistance in human leukemia HL-60 and adriamycin-resistant HL-60/ADR cells. HL-60/ADR cells showed significantly increased exosome production and release compared with parental chemosensitive HL-60 cells. In HL-60/ADR cells, increased SM synthase (SMS) activity reduced ceramide levels, although released exosomes exhibited a high ceramide ratio in both HL-60- and HL-60/ADR-derived exosomes. Overexpression of SMS2 but not SMS1 suppressed intracellular ceramide levels and accelerated exosome production and release in HL-60 cells. Notably, HL-60/ADR exosomes conferred cell proliferation and doxorubicin resistance properties to HL-60 cells. Finally, microRNA analysis in HL-60 and HL-60/ADR cells and exosomes showed that miR-484 elevation in HL-60/ADR cells and exosomes was associated with exosome-mediated cell proliferation. This suggests that intracellular ceramide metabolism by SMS2 regulates exosome production and release, leading to acquisition of drug resistance and enhanced cell proliferation in leukemia cells.

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.

Background: The protracted preclinical stage of Alzheimer’s disease (AD) provides the opportunity for early intervention to prevent the disease; however, the lack of minimally invasive and easily detectable biomarkers and their measurement technologies remain unresolved. Extracellular vesicles (EVs) are nanosized membrane vesicles released from a variety of cells and play important roles in cell–cell communication. Neuron-derived and ganglioside-enriched EVs capture amyloid-ß protein, a major AD agent, and transport it into glial cells for degradation; this suggests that EVs influence Aß accumulation in the brain. EV heterogeneity, however, requires the use of a highly sensitive technique for measuring specific EVs in biofluid. In this study, immuno-digital invasive cleavage assay (idICA) was developed for quantitating target-intact EVs. Methods: EVs were captured onto ganglioside GM1-specific cholera toxin B subunit (CTB)-conjugated magnetic beads and detected with a DNA oligonucleotide-labeled Aß antibody. Fluorescence signals for individual EVs were then counted using an invasive cleavage assay (ICA). This idICA examines the Aß-bound and GM1-containing EVs isolated from the culture supernatant of human APP-overexpressing N2a (APP-N2a) cells and APP transgenic mice sera. Results: The idICA quantitatively detected Aß-bound and GM1-containing EVs isolated from culture supernatants of APP-N2a cells and sera of AD model mice. The idICA levels of Aß-associated EVs in blood gradually increased from 3- to 12-month-old mice, corresponding to the progression of Aß accumulations in the brain of AD model mice. Conclusions: The present findings suggest that peripheral EVs harboring Aß and GM1 reflect Aß burden in mice. The idICA is a valuable tool for easy quantitative detection of EVs as an accessible biomarker for preclinical AD diagnosis.

Diet-derived bioactive lipids may help maintain cognitive resilience during the preclinical stage of Alzheimer's disease (AD). Konjac-derived glucosylceramide (kGlcCer), traditionally used for skin barrier support, has previously been shown to reduce brain amyloid accumulation in individuals with lower baseline amyloid burden. However, earlier studies used cognitive tests with limited sensitivity. The objective of this study was to investigate whether daily intake of kGlcCer improves cognitive performance and influences amyloid-related biomarkers in cognitively unimpaired older adults with subjective cognitive concerns. A 24-week randomized, double-blind, placebo-controlled, parallel-group trial. Forty cognitively unimpaired older adults aged 60-80 years who reported subjective decline in memory or cognitive function were enrolled and randomly assigned to study groups. Participants received placebo or kGlcCer at doses of 1.8, 3.6, or 5.4 mg/day for 24 weeks. Cognitive outcomes were assessed using the Wechsler Memory Scale-Revised (WMS-R) Logical Memory I/II, Wechsler Adult Intelligence Scale-Fourth Edition (WAIS-IV) Coding, and Standard Verbal Paired-Associate Learning Test (S-PA). Brain amyloid accumulation was estimated using plasma amyloid-β (Aβ) composite biomarker levels. In this exploratory pilot trial, kGlcCer intake was associated with changes in several cognitive measures, although consistent between-group superiority was not observed across all outcomes. The medium-dose group showed significantly greater improvement in WAIS-IV Coding performance compared with the placebo group. Although significant within-group improvements were observed in several measures in the high-dose group such as immediate and delayed verbal memory assessed by WMS-R Logical Memory I/II, as well as in unrelated word-pair learning on the S-PA, consistent placebo-adjusted between-group differences were not observed across these outcomes. Stratified analysis further revealed kGlcCer intake may be associated with favorable changes in amyloid-related biomarkers in high-dose participants with low baseline amyloid burden. No safety concerns related to kGlcCer intake were observed during the study period. These exploratory findings suggest that kGlcCer may be associated with changes in selected cognitive domains and amyloid-related biomarkers. However, the present pilot study was not powered to establish efficacy, and larger adequately powered randomized trials are required. UMIN000051463, date of registration: 07-03-2023.