Journal: Science advances

Article Title: ATP1A3 as a target for isolating neuron-specific extracellular vesicles from human brain and biofluids.

doi: 10.1126/sciadv.adi3647

Figure Lengend Snippet: Fig. 1. ATP1A3 is a neuron-specific EV marker candidate besides NCAM1 and L1CAM and associated with EVs in human brain tissues and biofluids at a single- particle level. (A) Experimental design for evaluating whether NDEV marker candidates (ATP1A3, NCAM1, and L1CAM) is associated with EVs. UC, ultracentrifugation; SU- GC, sucrose gradient centrifugation; SEC, size exclusion chromatography. (B) Transmission electron micrograph showing immunogold labeling of EVs isolated from iNeuron, brain, CSF, and plasma stained with anti-CD9 antibody (10 nm) and anti-ATP1A3, NCAM1, and L1CAM antibody (5 nm). Scale bars, 100 nm. (C) Representative fluorescent images of iNeuron-EV detected by fluorescent-conjugated antibodies (red: ATP1A3-AF647, NCAM1-AF647 or L1CAM-AF647; blue: CD9/81C/63-AF488) on Exoview chip CD63 capture spots. The round circle in magnified images masks costained particles with tetraspanins. Scale bars, 25 and 5 μm. (D) Percentages of ATP1A3, NCAM1, and L1CAM fluorescent particle counts relative to total particles in iNeuron derived EVs (n = 2 biological replicates) on Exoview chip tetraspanins capture spots. (E) Representative fluorescent images of brain- and CSF-EV detected by fluorescent-conjugated antibodies (green: ATP1A3-AF555; red: NCAM1-AF647 or L1CAM-AF647; blue: CD9/81/63-AF488) on Exoview chip CD9 capture spots. The round circle in magnified images masks co-stained particles with tetraspanins. Scale bars, 25 and 5 μm. (F) Percentages of ATP1A3, NCAM1, and L1CAM fluorescent particle counts relative to total particles in brain (n = 2) and CSF (n = 2) derived EVs on Exoview chip tetraspanins capture spots.

Article Snippet: In our experiment, EV antibodies cocktail (EV-TC-AB-01, NanoView Biosciences) containing AF488-conjugated anti-CD63, anti-CD81, and anti-CD9 antibodies, AF647-conjugated anti-ATP1A3 antibody (NBP299238AF647, Novus Biologicals), AF647-conjugated anti-NCAM1 antibody (catalog no. 563443, BD Biosciences), and AF647-conjugated anti-L1CAM antibody (catalog no. 564194, BD Biosciences) were used for detection of EV subpopulations.

Techniques: Marker, Single Particle, Gradient Centrifugation, Size-exclusion Chromatography, Transmission Assay, Labeling, Isolation, Clinical Proteomics, Staining, Derivative Assay

Journal: Science advances

Article Title: ATP1A3 as a target for isolating neuron-specific extracellular vesicles from human brain and biofluids.

doi: 10.1126/sciadv.adi3647

Figure Lengend Snippet: Fig. 2. Comparative analysis of NDEV candidate markers (ATP1A3, NCAM1, and L1CAM) expression in EVs by super-resolution microscopy. (A) Schematics showing the staining protocol of ATP1A3 and NCAM1 for EVs. (B) Representative super-resolution microscopy images showing the expression of ATP1A3 and NCAM1 in pan-tetraspanins (CD9/81/63) positive EVs at a single-particle level. Scale bars, 100 nm. (C) Pie charts showing the heterogeneous EV population with ATP1A3 and NCAM1 in different EV preparations. Particles with diameter less than 200 nm were quantified. (D) Schematics showing the staining protocol of ATP1A3 and L1CAM for EVs. (E) Representative super-resolution microscopy images showing the expression of ATP1A3 and L1CAM in pan-tetraspanins (CD9/81/63) positive EVs at a single- particle level. Scale bars, 100 nm. (F) Pie charts showing the heterogeneous EV population with ATP1A3 and L1CAM in different EV preparations. Particles with diameter less than 200 nm were quantified.

Article Snippet: In our experiment, EV antibodies cocktail (EV-TC-AB-01, NanoView Biosciences) containing AF488-conjugated anti-CD63, anti-CD81, and anti-CD9 antibodies, AF647-conjugated anti-ATP1A3 antibody (NBP299238AF647, Novus Biologicals), AF647-conjugated anti-NCAM1 antibody (catalog no. 563443, BD Biosciences), and AF647-conjugated anti-L1CAM antibody (catalog no. 564194, BD Biosciences) were used for detection of EV subpopulations.

Techniques: Expressing, Super-Resolution Microscopy, Staining, Single Particle

Journal: Science advances

Article Title: ATP1A3 as a target for isolating neuron-specific extracellular vesicles from human brain and biofluids.

doi: 10.1126/sciadv.adi3647

Figure Lengend Snippet: Fig. 3. ATP1A3+ EV immunoprecipitated from human brain tissues displays higher neuronal cell type specificity. (A) Workflow for characterizing ATP1A3+, NCAM1+, and L1CAM+ EVs immunoprecipitated from brain. (B) Western blot images of 5% total EVs (Input), supernatant (S.P), and immunoprecipitated EVs (A, ATP1A3; L, L1CAM; N, NCAM1) isolated from control brains (n = 3) and probed for neuron and glia markers. Equivalent proteins from brain-EVs were used for immuno- capturing ATP1A3, L1CAM, and NCAM1. IB, immunoblotting antibody (C) Principal components analysis (PCA). The EV proteome of total brain-EV and immunoprecip- itated EVs were measured in triplicate and classified. (D) Venn diagram showing the number of proteins differentially identified in total, ATP1A3+, L1CAM+, and NCAM1+

Article Snippet: In our experiment, EV antibodies cocktail (EV-TC-AB-01, NanoView Biosciences) containing AF488-conjugated anti-CD63, anti-CD81, and anti-CD9 antibodies, AF647-conjugated anti-ATP1A3 antibody (NBP299238AF647, Novus Biologicals), AF647-conjugated anti-NCAM1 antibody (catalog no. 563443, BD Biosciences), and AF647-conjugated anti-L1CAM antibody (catalog no. 564194, BD Biosciences) were used for detection of EV subpopulations.

Techniques: Immunoprecipitation, Western Blot, Isolation, Control

Journal: Science advances

Article Title: ATP1A3 as a target for isolating neuron-specific extracellular vesicles from human brain and biofluids.

doi: 10.1126/sciadv.adi3647

Figure Lengend Snippet: Fig. 4. ATP1A3+ EV analysis in human CSF and plasma. (A) Workflow for characterizing ATP1A3+ EVs immunoprecipitated from CSF. (B) Simoa quantification of AD related neuropathogenic proteins (Aβ42, Aβ40, T-tau, P-tau181, and SNAP25) in ATP1A3+ CSF-EVs from healthy controls (n = 3) and AD (n = 3). Data are presented by box and whisker plot which shows the median and the 25th to 75th percentiles. (C) Aβ42/Aβ40 ratio in CSF, total CSF-EV, and ATP1A3 immunoprecipitation (IP) CSF-EV samples among AD group. (D) Workflow explaining single-particle analysis of Aβ+ population in ATP1A3+ plasma-EV to distinguish AD from healthy controls (CTL) and mild cognitive impairment (MCI). (E) Representative super-resolution microscopy images showing plasma-derived Aβ+ ATP1A3+ single EV among control, MCI, and AD groups [green: FITC-CD9/81/63, red: Alexa Flour 555-ATP1A3, gray: Alexa Flour 647-Aβ (clone 4G8)]. Scale bars, 100 nm. (F) Percentage of Aβ+ population in ATP1A3+ plasma-EV among control, MCI, and AD groups (n = 10 per group). (G) Correlations of Aβ+ population in ATP1A3+ plasma-EV with clinical Braak stage, Mini- Mental State Examination (MMSE), and Dementia Rating Scale (DRS) scores in combined control (n = 7), MCI (n = 10), and AD (n = 10) samples. The dashed line indicates 95% confidence band of the best-fit line. Pearson correlation coefficients are shown. (H) Quantification of Aβ42/Aβ40 level in total plasma samples from CTL (n = 9), MCI (n = 10), and AD (n = 10). (I to J) Receiver-operating characteristics (ROC) curves displaying the performance of Aβ+ population in ATP1A3+ plasma-EV, plasma Aβ40, Aβ42, Aβ42/Aβ40 ratio, and T-tau to distinguish (I) AD from CTL and (J) AD from MCI. T-tau, total tau; AUC, area under the curve; CI, confidence interval; ns, no significance; *P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: In our experiment, EV antibodies cocktail (EV-TC-AB-01, NanoView Biosciences) containing AF488-conjugated anti-CD63, anti-CD81, and anti-CD9 antibodies, AF647-conjugated anti-ATP1A3 antibody (NBP299238AF647, Novus Biologicals), AF647-conjugated anti-NCAM1 antibody (catalog no. 563443, BD Biosciences), and AF647-conjugated anti-L1CAM antibody (catalog no. 564194, BD Biosciences) were used for detection of EV subpopulations.

Techniques: Clinical Proteomics, Immunoprecipitation, Whisker Assay, Single Particle, Super-Resolution Microscopy, Derivative Assay, Control