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Eitan et al. Extracell Vesicles Circ Nucleic Acids 2023;4:133-150 https://dx.doi.org/10.20517/evcna.2023.13 Page 145

Figure 4. NDEVs and their source plasma samples were used to measure BDNF and proBDNF in early AD and control donors. (A)
Measurements in the BioIVT cohort showed high enrichment of proBDNF levels in NDEVs (P < 0.001) compared to unprocessed
plasma. Moreover, we observed lower levels of NDEVs-associated proBDNF in early AD compared to control individuals (P = 0.002),
while no difference was observed in unprocessed plasma. (B) BDNF was 2.6-fold lower in NDEVs than in plasma (P < 0.01) and did not
vary between AD and controls. (C) ProBDNF measurements in two independently stored aliquots from 20 samples showed a
2
moderately strong correlation (R =0.7, P < 0.0001). (D, E) NDEVs-associated proBDNF was measured in two additional cohorts from
NIA and PMED; the decrease in NDEVs-associated proBDNF in early AD compared to control individuals was reproduced in the NIA
cohort (P = 0.001), while the PMED cohort generated a similar trend that did not reach statistical significance (P = 0.09), potentially
due to an insufficient number of control samples.

achieved significant enrichment of neuronal-specific cargo (proteins and RNA) through ExoSORT with
GAP43 and NLGN3 antibodies.

Our method specificity for EV capture was corroborated by transmission electron microscopy, nanoparticle
tracking analysis, western blotting, and ELISA measurement of EV-specific and non-EV (negative)
markers [26,44,45] , as well as by unbiased proteomic and lipidomic analyses, according to the MISEV 2018
[19]
guidelines . Interestingly, we found that contamination of NDEVs by ApoA was stronger than albumin,
consistent with reports regarding EV and HDL/LDL interactions . Measurement of multiple neuron-
[26]
specific proteins and mRNAs showed dramatic enrichment in NDEVs isolated by ExoSORT compared to
[27]
a procedural control (non-specific IgG), strongly supporting the specificity of ExoSORT towards neuronal
EVs.

Since most blood mRNA species are enclosed in EVs [28,46,47] , we compared NDEVs and plasma levels of
neuronal mRNAs and mRNAs originating from highly abundant blood components (erythrocytes and
platelets) and the liver. While non-neuronal mRNA levels in NDEVs were reduced by nearly 20-fold
compared to plasma, neuronal mRNA levels were similar between NDEVs and plasma, suggesting high
capture specificity and efficiency. The high efficiency of ExoSORT was further confirmed by ascertaining