TARGETED METABOLOMIC PROFILING OF ASTROCYTE LYSATES, CONDITIONED MEDIA AND EXTRACELLULAR VESICLES

Astrocytes are key regulators of nervous system homeostasis and synaptic function via metabolic support and intercellular signaling and like other cells release extracellular vesicles (Evs) that can further mediate intercellular communication by transferring bioactive cargo. This vesicle-based transfer raises the question of how various metabolites are compartmentalized across intracellular, soluble extracellular, and EVs-associated pools, and whether this distribution shifts under pathological conditions. Our goal was to characterize the distribution of a targeted metabolite panel using a human iPSC-derived astrocyte model. We analyzed excitatory, inhibitory and additional proteinogenic amino acids, kynurenine, acetylcholine, adenosine and polyamines, in astrocyte lysates, conditioned media, and astrocyte-derived EVs by liquid chromatography-tandem mass spectrometry. We identified an enrichment of several analytes in conditioned media relative to lysates (e.g. glutamine, glycine, tryptophan, tyrosine), whereas others were more lysate-associated (e.g., glutamate, spermine). Across shared compounds, EVs exhibited lower distribution of spermidine, tryptophan and tyrosine compared to conditioned media, while glutamate and aspartate were less reduced. Kynurenine was detected in conditioned media but not in EVs, and adenosine was detected in lysates and EVs but not in the media, suggesting compartment-dependent metabolite distributions. These findings can provide a basis for future studies using neuropsychiatric disease-elevant models to assess shifts in extracellular and vesicle-associated metabolomic patterns. This work has been funded by a grant from the Programme Johannes Amos Comenius under the Ministry of Education, Youth and Sports of the Czech Republic CZ.02.01.01/00/23_020/0008560.