Cargo molecules in plasma neural-derived extracellular vesicles reveal pathological signatures of primary open-angle glaucoma

Molecular biomarkers for glaucoma remain elusive. Neural-derived extracellular vesicles (NDEVs) offer diagnostic potential for neurodegenerative disorders. The aim of this study was to isolate NDEVs from the peripheral blood of patients with primary open-angle glaucoma (POAG) and investigate molecular alterations.

NDEVs were immunoprecipitated using specific antibodies against L1CAM and GLAST and characterized using transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA), and western blotting. ELISA was used to validate the enrichment of CNS markers and retina-specific proteins in the NDEV subtypes. The diagnostic efficacy of tau and GFAP in cell-free plasma, total EVs, and NDEVs for POAG was compared via ELISAs. MicroRNA (miRNA) sequencing was performed using NDEVs. A chronic ocular hypertension (COH) mouse model was established. The retinal expression of NDEV-derived differentially expressed miRNAs (DE-miRs) was verified by real-time PCR. Regulatory mechanisms were explored by integrating retinal mRNA sequencing with target gene prediction.

Immunoelectron microscopic analysis confirmed that GLAST proteins were localized in the EV membrane. Tau and GFAP levels were significantly elevated in the NDEVs of patients with POAG but not in cell-free plasma or total EVs, indicating the superior diagnostic value of these parameters for POAG. The target genes of DE-miRs in L1CAM⁺ EVs were biased towards neuronal functions, whereas those in GLAST⁺ EVs were biased towards glial inflammation. In the retinas of COH mice, the expression of miR-140-3p and miR-183-5p was significantly upregulated, which was consistent with the results of the human plasma NDEV sequencing. The target genes of the DE-miRs were intersected with differentially expressed genes (DEGs) in the retina, and the overlapping genes were enriched in axon development and glial cell activation pathways.

In addition to L1CAM, GLAST can be used as a membrane surface marker protein for the isolation of NDEVs. This study provides a novel strategy for the discovery of noninvasive biomarkers for neurological diseases and elucidation of their potential molecular mechanisms.