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L1CAM is not Associated with Extracellular Vesicles in Human Cerebrospinal Fluid or Plasma

By Maia Norman, Dmitry Ter-Ovanesyan, Wendy Trieu, Roey Lazarovits, Emma J.K. Kowal, Ju Hyun Lee, Alice Chen-Plotkin, Aviv Regev, George Church, David R. Walt

Posted 13 Aug 2020
bioRxiv DOI: 10.1101/2020.08.12.247833

Neuron-derived extracellular vesicles (NDEVs) present a tremendous opportunity to learn about the biochemistry of brain cells in living patients. L1CAM is a transmembrane protein expressed in neurons that is presumed to be found on NDEVs in human biofluids. Previous studies have used L1CAM immuno-isolation from human plasma to isolate NDEVs for neurodegenerative disease diagnostics. We developed a panel of ultrasensitive Single Molecule Array (Simoa) assays for known EV markers, as well as L1CAM, and applied it to study EVs in human plasma and cerebrospinal fluid (CSF). We fractionated plasma and CSF by size exclusion chromatography (SEC) and density gradient centrifugation (DGC) to separate EVs from free proteins. We observed that L1CAM did not elute in the EV fractions, but rather eluted in the free protein fractions. We found that L1CAM is present as a free protein in human plasma and CSF, possibly due to proteolytic cleavage and/or alternative splicing. We further demonstrate that the isoforms found in CSF and plasma are different. These data collectively establish that L1CAM in plasma is not EV associated and should therefore not be used for NDEV isolation. Importantly, the framework and tools described herein will allow for evaluation of other potential candidate markers for isolation of NDEVs. ### Competing Interest Statement DRW is a founder and equity holder of Quanterix. AR is a SAB member of ThermoFisher Scientific, Neogene Therapeutics, Asimov and Syros Pharmaceuticals. AR is a cofounder of and equity holder in Celsius Therapeutics and an equity holder in Immunitas. From August 1, 2020, AR is an employee of Genentech. GMC is a founder, consultant, or advisory board member to companies listed here: http://arep.med.harvard.edu/gmc/tech.html The authors have filed intellectual property on methods for EV analysis and isolation.

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