Lipidomic and proteomic insights from extracellular vesicles in the postmortem dorsolateral prefrontal cortex reveal...

Substance use disorder (SUD) significantly increases the risk of neurotoxicity, inflammation, oxidative stress, and impaired neuroplasticity. The activation of inflammatory pathways by substances may lead to reactive astrogliosis and chronic neuroinflammation, potentially mediated by the release of extracellular particles (EPs), such as extracellular condensates (ECs) and extracellular vesicles (EVs). These particles, which reflect the physiological, pathophysiological, and metabolic states of their cells of origin, might carry molecular signatures indicative of SUD. In particular, our study investigated neuroinflammatory signatures in SUD patients by isolating EVs from the dorsolateral prefrontal cortex (dlPFC) Brodmann's area 9 (BA9) from postmortem subjects. We isolated BA9-derived EVs from postmortem brain tissues of eight individuals (controls: n = 4, SUD: n = 4). The physical properties (concentration, size, zeta potential, morphology) of the EVs were analyzed, and the EVs were subjected to integrative multiomics analysis to profile the lipidomic and proteomic characteristics. We assessed the interactions and bioactivity of EVs by evaluating their uptake by glial cells. We further assessed the effects of EVs on complement mRNA expression in glial cells and on microglial migration. No significant differences in EV concentration, size, zeta potential, or surface markers were observed between the SUD group and the control group. However, lipidomic analysis revealed significant enrichment of glycerophosphoinositol bisphosphate (PIP2) in SUD-derived EVs. Proteomic analysis revealed the downregulation of SERPINB12, ACYP2, CAMK1D, DSC1, and FLNB and the upregulation of C4A, C3, and ALB in SUD-derived EVs. Gene Ontology (GO) and protein‒protein interactome analyses revealed functions associated with the identified proteins, such as cell motility, focal adhesion, and acute phase response signaling. Both control and SUD-derived EVs increased C3 and C4 mRNA expression in microglia, but only SUD-derived EVs upregulated these genes in astrocytes. SUD-EVs also significantly enhanced microglial migration in a wound healing assay. This study successfully isolated EVs from postmortem brains and used a multiomics approach to identify EV-associated lipids and proteins in SUD. Elevated C3 and C4 in SUD-derived EVs and the distinct effects of EVs on glial cells suggest a crucial role for these cells in acute phase response signaling and neuroinflammation.