Exploratory Proteomics of Brain Extracellular Vesicle Enriched Preparations After LPS Challenge and miRNA Associated Vesicle Administration

We used brain extracellular vesicle (EV) proteomics to identify protein responses to acute neuroinflammation and an EV-based microRNA intervention. Rats received vehicle, intracerebroventricular lipopolysaccharide (LPS), or LPS followed by an intravenous EV formulation containing anti-inflammatory microRNAs (miRNAs). Brains were collected 24 h after LPS or vehicle administration, and three independently processed animal-derived EV-enriched preparations per group were analyzed by label-free proteomics. Across all nine samples, 953 protein groups had complete normalized abundance measurements. Compared with vehicle controls, LPS exposure was associated with 227 exploratory protein changes, comprising 95 increases and 132 decreases at nominal P = log2(1.5). Increased proteins were enriched for translation-related annotations, whereas decreased proteins were enriched for plasma-membrane annotations. These LPS-associated changes did not remain significant after Benjamini-Hochberg correction at a false discovery rate (FDR) threshold of q < 0.05. Following treatment, 119 of the 227 candidates were closer to control abundance on the log2 scale; 23 candidates also showed nominal treatment-versus-LPS differences. Lamb2 and Ptgfrn were the two proteins that differed significantly between treated and LPS-only animals after FDR correction, identifying them as priorities for further validation. Nevertheless, 174 proteins differed significantly between treated animals and vehicle controls, and analysis across the measured proteins did not demonstrate an overall shift toward control values. Thus, the intervention was associated with specific protein responses, but treated animals retained substantial differences from controls. This study identifies candidate markers for investigating treatment responses in brain EV-enriched preparations and demonstrates the value of examining individual proteins alongside broader proteomic changes. Independent validation is needed to determine their functional relevance and distinguish miRNA-specific effects from contributions of the administered EV carrier.