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Plasma Extracellular Vesicles Enriched for Neuronal Origin: A Potential Window into Brain Pathologic Processes

Mustapic, Maja, Eitan, Erez, Werner, John K., Berkowitz, Sean T., Lazaropoulos, Michael P., Tran, Joyce, Goetzl, Edward J., Kapogiannis, Dimitrios·2017·Frontiers in Neuroscience
DOI 10.3389/fnins.2017.00278PMC PMC5439289
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Figure 1 Graphical workflow of the neuronal enrichment protocol. W/i signifies the addition of protease and phosphatase inhibitors containing 2–3x the concentration recommended by the manufacturer.
Figure 1 Graphical workflow of the neuronal enrichment protocol. W/i signifies the addition of protease and phosphatase inhibitors containing 2–3x the concentration recommended by the manufacturer.CC BY · PubMed Central
Figure 2 Neuronal EVs are found in the circulation. (A) Total EVs and L1CAM+ EVs were isolated from plasma of nine healthy volunteers and their size distribution was examined by NTA using Nanosight-NS500. Figure depicts concentration after 1:1,000 dilution for Total EVs and 1:200 dilution for L1CAM+ EVs; actual concentrations are depicted in (B). (B) The graph shows the actual concentration of total EVs and L1CAM+ and CD81+ plasma EVs immunoprecipitated from plasma of nine healthy volunteers after adjusting for dilution. The percentages in the graph represent the ratio over total EVs. (C) Western blot image shows enrichment of neuronal markers (L1CAM, MAP-2, N-enolase, p-Tau235, and TUJ) in L1CAM+ EVs when compared to total EVs from a single healthy control. CD9 is a common exosomal marker present in EVs but not in the mouse brain lysate used as a positive control. An equivalent amount of EVs was loaded on the gel by adjusting the dilution of the isolates according to the EV concentration determined by NTA. (D) GFP levels evaluated by FACS; L1CAM+ EVs were isolated from 300 μl plasma derived from Nestin-GFP transgenic or WT mice. The EVs were conjugated with the beads and the levels of GFP were evaluated by FACS analysis. The results show the percentages of beads-antibody-EV complex that contained GFP above the detection threshold. (E) GFP levels evaluated by fluorescence; comparison between the levels of GFP in EVs in the samples described in (D) were measured by plate reader at excitation of 485 nm and emission 515 nm.
Figure 2 Neuronal EVs are found in the circulation. (A) Total EVs and L1CAM+ EVs were isolated from plasma of nine healthy volunteers and their size distribution was examined by NTA using Nanosight-NS500. Figure depicts concentration after 1:1,000 dilution for Total EVs and 1:200 dilution for L1CAM+ EVs; actual concentrations are depicted in (B). (B) The graph shows the actual concentration of total EVs and L1CAM+ and CD81+ plasma EVs immunoprecipitated from plasma of nine healthy volunteers after adjusting for dilution. The percentages in the graph represent the ratio over total EVs. (C) Western blot image shows enrichment of neuronal markers (L1CAM, MAP-2, N-enolase, p-Tau235, and TUJ) in L1CAM+ EVs when compared to total EVs from a single healthy control. CD9 is a common exosomal marker present in EVs but not in the mouse brain lysate used as a positive control. An equivalent amount of EVs was loaded on the gel by adjusting the dilution of the isolates according to the EV concentration determined by NTA. (D) GFP levels evaluated by FACS; L1CAM+ EVs were isolated from 300 μl plasma derived from Nestin-GFP transgenic or WT mice. The EVs were conjugated with the beads and the levels of GFP were evaluated by FACS analysis. The results show the percentages of beads-antibody-EV complex that contained GFP above the detection threshold. (E) GFP levels evaluated by fluorescence; comparison between the levels of GFP in EVs in the samples described in (D) were measured by plate reader at excitation of 485 nm and emission 515 nm.CC BY · PubMed Central
Figure 3 Immuno Electron Microscopy images of L1CAM+ and CD81+ EVs. L1CAM+ or CD81+EVs were incubated with primary human anti-L1CAM or CD81 antibody followed by a secondary antibody conjugated with 6 nm gold particle. Scale bar = 100 nm, microscope settings; 120 kV, magnification 200,000x except the L1CAM+ labeled with anti-L1CAM (top left) which used a magnification of 160000x.
Figure 3 Immuno Electron Microscopy images of L1CAM+ and CD81+ EVs. L1CAM+ or CD81+EVs were incubated with primary human anti-L1CAM or CD81 antibody followed by a secondary antibody conjugated with 6 nm gold particle. Scale bar = 100 nm, microscope settings; 120 kV, magnification 200,000x except the L1CAM+ labeled with anti-L1CAM (top left) which used a magnification of 160000x.CC BY · PubMed Central
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.
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.CC BY · PubMed Central
Figure 5 L1CAM+ EVs offer a higher detection level for p-tau, BDNF and pro-BDNF over plasma, serum and total EVs. For p-tau comparisons, total EVs were isolated from four plasma and serum samples from healthy volunteers followed by L1CAM immunoprecipitation. The levels of p-tau-Thr181 (A) and p-tau-Thr231 (B) are presented in the graph in L1CAM+ EVs, total EVs, plasma, serum, and in comparison, to the background signal (blank). Column bars represent the mean of four samples, error bars represent SEM. For BDNF and proBDNF comparisons, total EVs were isolated from 20 plasma samples from healthy volunteers followed by L1CAM immunoprecipitation. BDNF levels (C) are different depending on the type of fluid tested [F(2, 57) = 6.868, p = 0.002]; its levels are higher in L1CAM + EVs compared to plasma (p = 0.001) and total EVs (p = 0.016), whereas its levels in total EVs were no different than plasma (p = 0.254). Similarly, proBDNF levels (D) are dependent on the type of fluid tested [F(2, 57) = 4.41, p = 0.017]; its levels are higher in L1CAM + EVs compared to plasma (p = 0.007) and total EVs (p = 0.026), whereas its levels in total EVs were no different than plasma (p = 0.628).
Figure 5 L1CAM+ EVs offer a higher detection level for p-tau, BDNF and pro-BDNF over plasma, serum and total EVs. For p-tau comparisons, total EVs were isolated from four plasma and serum samples from healthy volunteers followed by L1CAM immunoprecipitation. The levels of p-tau-Thr181 (A) and p-tau-Thr231 (B) are presented in the graph in L1CAM+ EVs, total EVs, plasma, serum, and in comparison, to the background signal (blank). Column bars represent the mean of four samples, error bars represent SEM. For BDNF and proBDNF comparisons, total EVs were isolated from 20 plasma samples from healthy volunteers followed by L1CAM immunoprecipitation. BDNF levels (C) are different depending on the type of fluid tested [F(2, 57) = 6.868, p = 0.002]; its levels are higher in L1CAM + EVs compared to plasma (p = 0.001) and total EVs (p = 0.016), whereas its levels in total EVs were no different than plasma (p = 0.254). Similarly, proBDNF levels (D) are dependent on the type of fluid tested [F(2, 57) = 4.41, p = 0.017]; its levels are higher in L1CAM + EVs compared to plasma (p = 0.007) and total EVs (p = 0.026), whereas its levels in total EVs were no different than plasma (p = 0.628).CC BY · PubMed Central
Figure 6 L1CAM+ EVs contain a distinct and interesting protein signature. Total EVs were isolated from plasma from four healthy volunteers and subsequently immunoprecipitated with L1CAM or EpCAM antibodies. The samples from the four subjects were then pooled to generate a sufficient amount of protein for five membrane antibody arrays [Human Obesity Antibody Array (A), Human Phospho-Kinase Antibody Array (B), MAPK Antibody Array (C), Human Kidney Biomarker Antibody Array (D), and Human Apoptosis Antibody Array (E)]. An equivalent amount of protein per sample type was loaded to each spot. The levels of different proteins were calculated by densitometric analysis and normalized to the reference proteins present on each membrane. Results are depicted as heatmaps ranging from zero to maximum value and scaled according to the color bars.
Figure 6 L1CAM+ EVs contain a distinct and interesting protein signature. Total EVs were isolated from plasma from four healthy volunteers and subsequently immunoprecipitated with L1CAM or EpCAM antibodies. The samples from the four subjects were then pooled to generate a sufficient amount of protein for five membrane antibody arrays [Human Obesity Antibody Array (A), Human Phospho-Kinase Antibody Array (B), MAPK Antibody Array (C), Human Kidney Biomarker Antibody Array (D), and Human Apoptosis Antibody Array (E)]. An equivalent amount of protein per sample type was loaded to each spot. The levels of different proteins were calculated by densitometric analysis and normalized to the reference proteins present on each membrane. Results are depicted as heatmaps ranging from zero to maximum value and scaled according to the color bars.CC BY · PubMed Central

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Authors
Mustapic, M., Eitan, E., Werner, J. K., Berkowitz, S. T., Lazaropoulos, M. P., Tran, J., Goetzl, E. J., & Kapogiannis, D.
Venue
Frontiers in Neuroscience
Publisher
Frontiers Media SA
Published
2017-05-22
Volume
11
ISSN
1662-453X
DOI
10.3389/fnins.2017.00278
PMC
PMC5439289
Cited by
441
References
13

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