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

ABSTRACT: Neuron-derived extracellular vesicles (NDEVs) are potential biomarkers of neurological diseases although their reliable molecular target is not well established. Here, we demonstrate that ATPase Na⁺/K⁺ transporting subunit alpha 3 (ATP1A3) is abundantly expressed in extracellular vesicles (EVs) isolated from induced human neuron, brain, cerebrospinal fluid, and plasma in comparison with the presumed NDEV markers NCAM1 and L1CAM by using super-resolution microscopy and biochemical assessments. Proteomic analysis of immunoprecipitated ATP1A3⁺ brain-derived EVs shows higher enrichment of synaptic markers and cargo proteins relevant to Alzheimer's disease (AD) compared to NCAM1⁺ or LICAM⁺ EVs. Single particle analysis shows the elevated amyloid-β positivity in ATP1A3⁺ EVs from AD plasma, providing better diagnostic prediction of AD over other plasma biomarkers. Thus, ATP1A3 is a reliable target to isolate NDEV from biofluids for diagnostic research.

SUBMITTER: You Y

PROVIDER: S-EPMC10881047 | biostudies-literature | 2023 Sep

REPOSITORIES: biostudies-literature

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

You Yang Y Zhang Zhengrong Z Sultana Nadia N Ericsson Maria M Martens Yuka A YA Sun Min M Kanekiyo Takahisa T Ikezu Seiko S Shaffer Scott A SA Ikezu Tsuneya T

Science advances 20230915 37

Neuron-derived extracellular vesicles (NDEVs) are potential biomarkers of neurological diseases although their reliable molecular target is not well established. Here, we demonstrate that ATPase Na<sup>+</sup>/K<sup>+</sup> transporting subunit alpha 3 (ATP1A3) is abundantly expressed in extracellular vesicles (EVs) isolated from induced human neuron, brain, cerebrospinal fluid, and plasma in comparison with the presumed NDEV markers NCAM1 and L1CAM by using super-resolution microscopy and bioch ...[more]

PMID: 37713494

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Project description:Extracellular vesicles that are derived from stem cells play an important role in the treatment of disease. To obtain high-quality small extracellular vesicles (sEVs), we optimized the culture conditions of human induced pluripotent stem cells (hiPSCs), the supernatant collection time, and sEVs extraction methods. Firstly, hiPSCs were cultured in extracellular vesicles-production medium (EVs-PM) containing different concentrations (0%, 0.25%, 0.5%, 2%, 5%, and 20%) of extracellular vesicle-depleted knockout serum replacement (ED-KSR), and the culture supernatants were collected continuously for 5 days. Then, the sEVs were isolated, followed by an evaluation of their characteristics. The survival rates of the hiPSCs lines that were cultured in EVs-PM containing 0.5% to 20% ED-KSR were not significantly different (P > 0.05). The survival rates of the hiPSCs in 0.5% ED-KSR after the culture supernatants were continuously collected for day 1, day 3, and day 5 were not statistically significant (P > 0.05). After 5 days of continuous collection of the supernatant, the hiPSCs expressed some pluripotent markers, while SSEA4 and TRA-1-60 expression changed gradually. The sEVs that were extracted by the two methods were all 50-200 nm, double-layered and oval or round cellular vesicles and expressed the marker proteins CD63, TSG101, and HSP70. The characteristics of sEVs extracted on day 1, day 3, and day 5 were almost identical on morphology, size and the relative quantity of annexin V-positive subpopulations. The PKH67 staining showed that the sEVs could be endocytosed by HepG2 cells and aggregated in the cytoplasm. The proliferation experiments showed that the sEVs can promote cell proliferation. In Conclusion, the 0.5% ED-KSR is the optimal concentration, and that the hiPSCs culture supernatant can be continuously collected for 5 days while maintaining high cell viability and some pluripotent characteristics. Both of the methods extraction can be used to obtain biologically active sEVs.

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

Publications

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

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