Single-extracellular vesicle (EV) analyses validate the use of L1 Cell Adhesion Molecule (L1CAM) as a reliable biomarker of neuron-derived EVs

Abstract

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% to 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.

Keywords: Alzheimer's disease; L1CAM; blood biomarkers; extracellular vesicles; neuron‐derived extracellular vesicles.

FIGURE 1

High‐resolution fluorescent confocal microscopy and nanoscale multiplex flow cytometry analysis detect L1CAM in single neuron‐derived EVs. (a) Diagram of the differential ultracentrifugation methodology used for the isolation of EVs from the conditioned media of rat hippocampal neurons and human iNeurons (namely, rat NDEVS and human iNDEVs, respectively) (created with BioRender; agreement number: MF23W0TRTS). The expression of L1CAM and additional neuronal markers in cultured neurons is demonstrated by immunocytochemistry (nuclei visualised using DAPI in blue; scale bars: 20 µm; scale bar of box inset: 10 µm). (b) Histogram of EV concentration as function of particle diameter from nanoparticle tracking analysis (NTA) of rat NDEVs, human iNDEVs and their respective vehicles (cell media subjected to the same EV isolation methods). (c) Immunoblots of rat and human EV and neuronal lysates showing the expression of full‐length L1CAM (asterisk) and products of lower molecular weight (arrowhead) (using anti‐L1CAM antibody clone EPR18750 under reducing conditions; Figure S2), as well as canonical neuronal (β‐III‐tubulin) and EV (CD81, Alix and Flotillin‐1) markers. (d and e) High resolution fluorescent confocal microscopy of rat NDEVs (d) and human iNDEVs (e) showing the co‐immunolabelling of L1CAM and EV markers in single particles with nano‐scale sizes confirmed by the detection of FITC‐positive beads with sizes ranging from 100 to 300 nm (scale bars: 500 nm). Images of selected nanoparticles were captured at a higher magnification and are shown in box insets (scale bars: 200 nm). High‐sensitivity nanoscale multiplex flow cytometry analysis (FCA) (f–o). Dot plots show the violet size scatter (vSSC) in function of the fluorescent signal of rat (f–j) and human (k–o) NDEV samples co‐labelled with the fluorescent EV marker BSE (h and l) and fluorescent antibodies against L1CAM (i and m). Human iNDEVs were also labelled with fluorescent antibodies against either tetraspanins (n) or VAMP2 (o). Gates enclosing events positive for a given marker were designated based on the background signal of negative controls (f, g and k). The frequency of events (events/sec) in analysed samples did not result in coinciding events as confirmed by swarming experiments (Figure S4a,b). Dot plots (n) and (o) include the percentage of L1CAM‐positive events double‐positive for tetraspanins and VAMP2. A size range based on the vSSC of FITC‐tagged beads (Figure S3) is included on the right of each panel for the size comparison of events.

FIGURE 2

Detection of EV‐associated L1CAM in human plasma. On the top of (a), a diagram (created with BioRender; agreement number: JD23W0UDJ4) illustrates the (partial) separation of EVs and soluble proteins from plasma using size exclusion chromatography (SEC). SEC performance was corroborated by the quantification of EVs and soluble plasma proteins in collected fractions using FCA of BSE+ EVs, BCA total protein assay, albumin ELISA (bottom panel in a) and interferometry nanoparticle analysis by ExoView^® (b and c). In (b) and (c), bar graphs show the number and size of tetraspanin‐positive EVs (based on the interferometry signal of particles captured by anti‐CD9, ‐CD63 or ‐CD81 antibodies) in equal volumes of SEC fractions. The signal from particles captured with an isotype control (mIgG) was subtracted from that of tetraspanin+ particles to account for the non‐specific binding of EVs to the ExoView^® chip. (d), Simoa^® assays for the detection of L1CAM in EVs from blood are illustrated (created with BioRender; agreement number: EN23W0UMNQ). In (e) and (f), bar graphs show the mean protein concentration of L1CAM or the average enzymes per bead (AEB) signal of L1CAM+ EVs as measured by L1CAM/L1CAM or L1CAM/pan‐tetraspanins Simoa^® assays, respectively. An inset in the L1CAM/L1CAM Simoa^® graph shows results with an adjusted y‐axis including the assay's LLoQ in red. Recombinant human L1CAM, iNDEVs and plasma EVs isolated by ultracentrifugation at 120,000 × g were used as positive controls. (g) Immuno‐TEM images showing the deposition of anti‐L1CAM antibody clone 5G3 (arrows) in nanoparticles from plasma (pooled SEC fractions 1–5) before (crude EVs) and after (eluate) L1CAM IP. For images of ‘Crude EVs’ and ‘L1CAM IP eluate’ samples showing L1CAM immunoreactivity, box insets on widefield images on the far left enclose the areas magnified in the centre panels which are followed by images on the right corresponding to replicate TEM images of the same samples. Images of the ‘L1CAM IP eluate’ sample incubated with secondary antibody (2^ry AB) alone on the far right assess non‐specific binding. Scale bars, 100 nm. (h) A bar graph of the AEB signal from the L1CAM/pan‐tetraspanins Simoa^® of EVs isolated from plasma via ExoQuick^® sedimentation before (crude EVs) and after (eluate) L1CAM IP. The low signal obtained by the supernatant collected after sedimentation of EVs (EV‐depleted plasma) suggests the EV‐specificity of the L1CAM/pan‐tetraspanins Simoa^® assay. The abolition of the signal with treatment of the L1CAM IP eluate with RIPA detergent further confirms the specificity of the signal for membrane enclosed tetraspanin+ particles. The AEB from free biotin accounts for the non‐specific signal of biotinylated IP antibodies in the L1CAM IP eluate. Statistical analysis: two‐way ANOVA; ****p < 0.0001. (i) High resolution fluorescent confocal microscopy of the L1CAM IP eluate showing the co‐immunolabelling of L1CAM and EV markers in single particles with nano‐scale sizes confirmed by the detection of FITC‐positive beads with sizes ranging from 100 to 300 nm (scale bars: 500 nm). Selected nanoparticles enclosed by box insets were captured at a higher magnification (scale bars: 200 nm). Graphs a–f and h average sample replicates from three different subjects. All experiments were repeated at least once using samples from additional donors with similar results.

FIGURE 3

Soluble L1CAM in blood does not binds to the surface of EVs. (a) A diagram (created with BioRender; agreement number: LX25DH5BU3) of the methodology followed to assess the binding of soluble L1CAM and ApoA1 in plasma to the surface of EVs in vitro. L1CAM‐negative EVs were isolated from the conditioned media of HEK‐293 cells, that naturally do not express L1CAM, via ultracentrifugation at 120,000 x g (step 1a) and labelled with the total EV marker BSE (step 1b). Flow cytometry analysis (FCA) confirmed the BSE labelling (vSSC vs. BSE dot plot on the left) and EV origin (vSSC vs. APC‐CD9/CD63/CD81 signal of BSE‐gated events on the right) of HEK‐derived EVs. Then, BSE‐labelled EVs were incubated with soluble plasma proteins from two human subjects separately (step 3), depleted of EVs by ExoQuick^® sedimentation (step 2), consisting of L1CAM and ApoA1 as confirmed by immunoblots (using L1CAM antibody clone EPR18750 targeting the ectodomain; refer to Figure S2 for target epitope). After incubation, BSE‐labelled EVs were purified from soluble plasma proteins by size exclusion chromatography (step 4) and subjected to fluorescent labelling with PE‐tagged anti‐L1CAM antibody (5G3 clone) or rabbit anti‐ApoA1 plus PE‐tagged anti‐rabbit antibodies for FCA (step 5). (b) Dot plots showing the vSSC versus PE signal of BSE‐gated events identified by FCA of BSE+ HEK‐derived EVs incubated with soluble plasma proteins from a human subject and labelled either with PE‐tagged anti‐L1CAM (red events, left), rabbit anti‐ApoA1 plus PE‐tagged anti‐rabbit antibodies (red events, right), or PE‐tagged anti‐rabbit antibody alone (green events, right). A PE gate designated based on the background signal of negative controls (unlabelled EVs and EVs labelled with secondary antibody) encloses events positive for L1CAM (left) or ApoA1 (right). (c) A bar graph indicates the mean percentage with standard error of HEK‐derived EVs positive for L1CAM or ApoA1 (PE gated) out of total EVs (BSE+ events), after incubation with soluble plasma proteins (red bars) or left untreated (blue bars). Error bars represent the standard error of the mean from values of EVs incubated with soluble plasma proteins from two human subjects.

FIGURE 4

Quantification of the percentage of L1CAM+ EVs in human plasma carrying canonical EV and neuronal markers. Flow cytometry analysis of crude EVs isolated from human plasma via ultracentrifugation (UC) at 120,000 × g and triple‐labelled with the fluorescent EV marker BSE, PE‐tagged anti‐L1CAM antibody (5G3 clone) and APC‐tagged antibodies targeting either pan‐tetraspanins (a cocktail of anti‐CD9, ‐CD63 and ‐CD81 antibodies), VAMP2 or β‐III‐tubulin. In (a) a dot plot shows the vSSC in function of the BSE fluorescent signal in crude plasma EVs from a human subject, either unlabelled (red events) or labelled with BSE before (black events) and after (beige events) treatment with 2% SDS. A colour‐coded size range based on the vSSC of FITC‐tagged beads (Figure S3) is included on the right for the size comparison of events. In (b) a dot plot overlays the vSSC versus BSE signal of PE‐ and APC‐tagged antibodies alone in PBS‐1X (red events), and crude plasma EVs from the same human subject as in (a), labelled either with BSE plus PE‐ and APC‐tagged antibodies (black events) or BSE only (light green events). A blue gate designated based on the background signal of negative controls (unlabelled EVs as well as PE‐ and APC‐tagged antibodies without EVs) encloses events positive for BSE. In (c) a dot plot overlays the vSSC versus PE signal of BSE‐gated events in crude plasma EVs from the same human subject as in (a) and (b) labelled either with BSE plus PE‐tagged anti‐L1CAM antibody (5G3 clone) (black events) or BSE only (light green events). A turquoise dashed line designated based on the background signal of negative controls (unlabelled EVs, EVs labelled with a PE‐tagged isotype control antibody, and PE‐tagged anti‐L1CAM antibody alone) gates events positive for L1CAM (events laying on the right of the dashed line). In (d) a histogram shows the number of L1CAM‐gated events in (c) (turquoise line) overlayed with results of EVs labelled with BSE plus PE‐tagged isotype control antibody (yellow line). The frequency of events in analysed samples (157 ± 55 events/s; n = 22 samples) did not resulted in coinciding events as confirmed by swarming experiments (Figure S4c,d). In (e) a bar graph shows the mean percentage of L1CAM‐gated events out of total BSE+ events in replicates (n = 4) from four different subjects (subtracted by the background signal from the isotype control antibody). In (f), (i) and (l), dot plots overlay the vSSC versus APC signal of BSE‐gated events in crude plasma EVs labelled either with BSE alone (light green events) or BSE plus PE‐tagged anti‐L1CAM antibody (representative vSSC vs. PE fluorescence shown in Figure 4c) and APC‐tagged anti‐CD9/CD63/CD81 (f), ‐VAMP2 (i) or ‐β‐III‐tubulin (l) antibodies (black events). Colour coded dashed lines designated based on the background signal of negative controls (unlabelled EVs, EVs labelled with an APC‐tagged isotype control antibody, and APC‐tagged antibodies without EVs) gates events positive for pan‐tetraspanins, VAMP2 or β‐III‐tubulin (events laying on the right of the dashed lines). In (g), (j) and (m), histograms show the number of APC‐gated events in f (purple), i (pink) and l (orange), respectively, overlayed with results of EVs labelled with BSE plus APC‐tagged isotype control antibody (yellow line). In (h), (k) and (n), dot plots identify the APC signal of L1CAM‐gated events (turquoise). In (o), a bar graph shows the mean percentage with standard error of L1CAM‐gated events positive for pan‐tetraspanins (purple), VAMP2 (pink) and β‐III‐tubulin (orange) out of total L1CAM‐gated events in individual samples from four different subjects. Similar results were obtained in crude plasma EVs isolated by SEC (data not shown).

FIGURE 5

L1CAM‐IP of human plasma results in the enrichment of L1CAM+ NDEVs. (a–d) Dot plots show the vSSC in function of the fluorescent signal of samples consisting of crude EVs from pooled SEC fractions 1–5 (a and b, turquoise events) or the L1 IP eluate of crude EVs (c and d, dark red events) triple‐labelled with the fluorescent EV marker BSE (shown in a and c), and fluorescent antibodies against L1CAM (shown in b and d) and either a mix of antibodies against CD9, CD63, CD81 (pan‐tetraspanins), VAMP2 or ASGR2 (shown in h–j, respectively). Yellow gates enclosing events positive for a given marker were designated based on the background signal of negative controls (green and black events). The frequency of events (events/s) in analysed samples, presented in graphs (a) and (c), did not result in coinciding events as confirmed by swarming experiments (Figure S4c,d). Dot plots (b) and (d) include the percentage of BSE‐gated events double‐positive for L1CAM. (e) Bar graph showing the average percentage of BSE‐gated events out of total events in the indicated samples from multiple subjects. (f) A histogram shows the abundance of L1CAM‐gated events in (b) and (d). (g) The average number of L1CAM‐gated events in the L1CAM IP eluate from multiple subjects is represented as fold‐change over crude EVs (n = 4 consisting of samples in (b) and (d), from the pooled plasma of two subjects, and samples from the plasma of three subjects assessed individually and shown in Figure S11a), using the eluate of an IP carried with an isotype control for the L1CAM IP antibody as a negative control. (h–j) Dot plots of events identified in the L1CAM IP eluate illustrate the percentage of L1CAM‐gated events, shown in colours, double‐positive for the indicated EV markers (enclosed by yellow gates). (k) Bar graph showing the average of (h)–(j) from multiple subjects. A colour‐coded size range based on the vSSC of FITC‐tagged beads (Figure S3a) is included on the right of (d), (h) and (j) for the size comparison of events. Data in (e), (g) and (k) represents the average of separate and pooled samples from multiple subjects (N = 3) processed and analysed in three individual experiments. Statistical analyses: one‐way ANOVA; *p < 0.05, ***p < 0.001, ****p < 0.0001; for e, the average of negative controls in grey was compared with the other experimental groups (l), A diagram illustrates a Luminex^® assay for the detection of EVs positive for GAP43, CD9 or CD63 in crude EVs isolated via SEC before and after L1CAM IP (created with BioRender; agreement number: ZJ23W0UUXG). Recorded mean fluorescence intensity (MFI) signals are graphed in (m) (statistical analysis: unpaired t‐test; *p < 0.05, **p < 0.005; average sample replicates from two different subjects with similar results obtained in a separate experiment using samples from additional donors).