Single-extracellular vesicle (EV) analyses validate the use of L1 Cell Adhesion Molecule (L1CAM) as a reliable biomarker of neuron-derived EVs
Description
Isolation of neuron-derived extracellular vesicles (NDEVs) with L1 Cell Adhesion Molecule (L1CAM)-specific antibodies has been widely used to identify blood biomarkers of CNS disorders. However, full methodological validation requires demonstration of L1CAM in individual NDEVs and lower levels or absence of L1CAM in individual EVs from other cells. Here, we used multiple single-EV techniques to establish the neuronal origin and determine the abundance of L1CAM-positive EVs in human blood. L1CAM epitopes of the ectodomain are shown to be co-expressed on single-EVs with the neuronal proteins β-III-tubulin, GAP43, and VAMP2, the levels of which increase in parallel with the enrichment of L1CAM-positive EVs. Levels of L1CAM-positive EVs carrying the neuronal proteins VAMP2 and β-III-tubulin range from 30-63%, in contrast to 0.8-3.9% of L1CAM-negative EVs. Plasma fluid-phase L1CAM does not bind to single-EVs. Our findings support the use of L1CAM as a target for isolating plasma NDEVs and leveraging their cargo to identify biomarkers reflecting neuronal function.
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Labelled EVs were detected with a CytoFLEX LX flow cytometer (Beckman Coulter) previously shown to resolve small EVs and polystyrene beads down to 100 nm in size combining violet (405 nm laser) side scatter (vSSC) and fluorescence detection. The general strategy used for the detection of fluorescently labeled EVs was fluorescence threshold triggering, setting the instrument to measure the vSSC and fluorescence intensities of events with a fluorescent signal over a threshold established using controls indicating background noise, as done in previous EV studies. The rationale for using fluorescence rather than particle scattering for event triggering was based on analyses of EVs stained with BSE showing that BSE-fluorescence threshold triggering provides a better signal to noise ratio over vSSC (data not shown). This choice was also supported by previous reports indicating that particulate matter in the sample buffer could trigger a scatter signal within the range of detected EVs, but such particles are less likely to exhibit autofluorescence. The nanoscale size resolution capability of CytoFLEX in our hands was confirmed using fluorescent nanoscale-sized beads ranging from 100-1300 nm (SPHERO™ Flow Cytometry Nano Fluorescent Size Standard Kit, yellow; Spherotech) and EVs from cpVenus-expressing HEK cells (courtesy of Dr. Xiaolu Cambronne) isolated via differential UC as previously described, identified via FITC-threshold triggering and vSSC detection. FITC-tagged nanobeads of 100, 220, 400, 880 and 1300 nm were analyzed individually before each experiment to assess the vSSC-based size resolution under set acquisition parameters.