Tetraspanins, GLAST and L1CAM Quantification in Single Extracellular Vesicles from Cerebrospinal Fluid and Serum of...

Objective: This study aimed to unravel the single tetraspanin pattern of extracellular vesicles (EVs), L1CAM⁺ and GLAST⁺ EV levels as diagnostic biomarkers to stratify people with multiple sclerosis (pwMS), specifically relapsing-remitting (RRMS) and primary progressive (PPMS). Methods: The ExoView platform was used to directly track single EVs using a clinically feasible volume of cerebrospinal fluid (CSF) and serum samples. This technology allowed us to examine the patterns of classical tetraspanin and quantify the levels of L1CAM and GLAST proteins, commonly used to immunoisolate putative neuron- and astrocyte-derived EVs. Results: The tetraspanin EV pattern does not allow us to differentiate RRMS, PPMS and non-MS donors neither in CSF nor serum, but this was associated with the type of biofluid. L1CAM⁺ and GLAST⁺ EVs showed a very low presence of tetraspanin proteins. Additionally, a significant decrease in the particle count of L1CAM⁺ EVs was detected in L1CAM-captured spots, and L1CAM⁺ and GLAST⁺ EVs decreased in GLAST-captured spots in the CSF from PPMS subjects compared to RRMS. Interestingly, only GLAST⁺ EVs exhibited a lower quantity in the CSF from PPMS compared to both MS and non-MS samples. Finally, GLAST⁺ EVs demonstrated a medium negative and significative correlation with GFAP levels-a biomarker of MS progression, astrocyte damage and neurodegenerative processes. Conclusions: ExoView technology could track neural EV biomarkers and be potentially useful in the diagnostic evaluation and follow-up of pwMS. GLAST⁺ EVs might provide insights into the etiology of PPMS and could offer small windows to elucidate the molecular mechanisms behind its clinical presentation.