fig1

Figure 1. NDEV isolation and characterization. The isolated NDEVs demonstrate key EV characteristics. (A) NDEVs were eluted from ExoSORT capture beads, fixed in 3% PFA, and visualized by transmission electron microscopy. Vesicular shape and size under 200 nm were observed. (B) The size distribution of eluted NDEVs was determined by nanoparticle tracking analysis. The NDEVs average diameter ± SEM was 127 ± 72 nm. (C) Western blot analysis (WES instrument) showed that NDEVs (N) contained enriched levels of typical EVs markers CD9 and FLOT1, and reduced levels of albumin, compared to unprocessed plasma (P). Mouse brain extract (B) was used as a control. Total protein loaded (ug) is shown for each lane. (D, E) Levels of albumin and ApoA1 were much lower in isolated NDEVs than in unprocessed plasma, as quantified by ELISA (R&D Systems, Cat. No. DY1455 and DY366405). The fractions of albumin and ApoA1 in NDEVs were 0.78 ± 0.2% and 4.05 ± 0.94% of the levels observed in unprocessed plasma, respectively (P < 0.0001 for both proteins). (F, G) The enrichment for neuronal material was ascertained by comparing neuron-specific proteins and RNA in plasma and NDEVs. (F) WES analysis showed that NDEVs (lanes labeled N) are enriched for neuronal markers NeuN and GluR2 compared to unprocessed plasma (lanes labeled P). Protein amounts loaded onto each lane are shown above. (G) mRNA encoding markers of EV origin from erythrocytes (hemoglobin, HBB), platelets (platelet factor 4, PF4), liver (albumin, ALB), and neurons (neurogranin, NRGN) were measured by QPCR in plasma and NDEVs. The resultant values were adjusted to the input volume. Note that there were lower levels of HBB, PF4, and ALB mRNA in NDEVs compared to plasma (1,4, 34, and 68-fold, respectively; P < 0.0001 for all comparisons). In contrast, NRGN mRNA levels were similar between NDEVs and plasma (P = 0.66).