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. 2017 May 22;11:278. doi: 10.3389/fnins.2017.00278

Figure 4.

Figure 4

L1CAM+ EVs are enriched for neuronal origin. (A) L1CAM+ and CD81+ plasma-derived EVs from five healthy volunteers (out of 10 showing similar data). Western blots of L1CAM+ EVs (L1+) are set adjacent to corresponding CD81+ EVs (81+) for each individual. The membrane was stained for L1CAM (top), neuron-specific enolase (NSE; middle), and EV marker CD9 (bottom). An equivalent amount of EVs were loaded on the gel by adjusting the dilution of the isolates according to the EV concentration determined by NTA. (B) Enrichment of neuronal markers in L1CAM+ EVs compared to CD81+ EVs by Western blots [L1CAM+ EVs (Blue, N = 10); CD81+ EVs (Red, N = 10)]. Enrichment is expressed as a fold difference in the ratio of L1CAM or NSE over CD9 signal. ImageJ was used to determine the signal intensity of each marker. A paired t-test was used to determine statistical differences between L1CAM+ and CD81+ EVs, error bars represent SEM of 10 subjects. Significance *p < 0.05, **p < 0.0001. (C) Enrichment of neuronal markers in L1CAM+ EVs compared to CD81+ EVs by ELISA for neuronal markers, NFL, NCAM, BDNF, proBDNF. (1) Fold difference in protein levels in L1CAM+ EVs to CD81+ EVs: L1CAM+ EVs contain 2.44 ± 0.56 (mean ± SEM) fold more NFL, 2.85 ± 1.19-fold more NCAM, and 2.16 ± 0.49-fold more proBDNF than CD81+ EVs (N = 10 healthy volunteers, measured in duplicate). L1CAM+ EVs contain amounts (0.94 ± 0.05) of BDNF similar to those of CD81+ EVs. (2) Fold difference in protein levels in L1CAM+ EVs to CD81+ EVs normalized to number of EV particles/ml measured by NTA. (3) Fold difference in protein levels in L1CAM+ EVs to CD81+ EVs normalized to TSG101 protein levels measured using custom electroluminescence assay. These results show that L1CAM+ EVs contain consistently and substantially higher levels of a range of neuronal proteins compared to total and control sub-populations.