Application of astrocyte-derived extracellular vesicles carrying Tau in the auxiliary diagnosis of progressive supranuclear palsy

Abstract

The present invention relates to the field of biomedical technology and discloses the use of astrocyte-derived extracellular vesicles (EVs) carrying Tau in the aided diagnosis of progressive supranuclear palsy (PSP). Using fluorescently labeled antibodies specific to Tau protein and anti-glutamate transporter 1 antibodies, the present invention accurately detects Tau-carrying EVs from astrocytes in plasma. By analyzing the concentration of Tau-carrying EVs in plasma, the present invention accurately differentiates patients with PSP from those with Parkinson's disease (PD) and healthy individuals. This provides a highly effective diagnostic biomarker and model for PSP, offering new empirical methods and technical support for the diagnosis and differentiation of PSP.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and in particular to the use of astrocyte-derived extracellular vesicles carrying Tau in the auxiliary diagnosis of progressive supranuclear palsy. Background Art

[0002] Progressive supranuclear palsy (PSP) is a common atypical Parkinson's syndrome characterized by abnormal aggregation of the protein Tau in neurons, oligodendrocytes, and astrocytes. Because Parkinson's disease (PD) and other atypical parkinsonian syndromes have multiple clinical subtypes and often overlapping symptoms, their diagnosis is complex, leading to a high rate of misdiagnosis. Therefore, reliable biomarkers are urgently needed to facilitate early diagnosis and effectively distinguish PSP from other neurodegenerative diseases.

[0003] In existing studies, abnormal tau protein deposition has been detected in a variety of biological fluids and peripheral tissues, including plasma, olfactory epithelial cells, oral epithelial cells, colon samples, and skin biopsies. Compared with tissue biopsies and other methods of obtaining plasma, as a routinely available clinical sample, it has significant advantages.

[0004] Current research uses methods such as immunocapture and enzyme-linked immunosorbent assays (ELISAs) to extract and analyze disease-associated extracellular vesicles (EVs) from plasma. However, these methods typically require large volumes of serum samples (typically 500 μL) and are complex to detect. Existing immunocapture and ELISA procedures are cumbersome and time-consuming, requiring multiple operations, which can lead to errors between steps and affect the reliability and reproducibility of the results. They also lack sensitivity and specificity, and lack non-invasive detection methods. Existing biological samples often require invasive procedures such as tissue biopsies, limiting their widespread application in routine medical care. Summary of the Invention

[0005] The present invention aims to provide the use of astrocyte-derived extracellular vesicles (EVs) carrying Tau in the aided diagnosis of progressive supranuclear palsy (PSP), addressing the aforementioned problems with the prior art. This method utilizes fluorescently labeled antibodies specific to Tau protein and anti-glutamate transporter 1 (G-T1) to accurately detect Tau-carrying EVs in plasma. By analyzing the concentration of Tau-carrying EVs in plasma, it can accurately distinguish patients with PSP from those with PD, as well as healthy individuals. This provides a highly effective diagnostic biomarker and model for PSP, offering new empirical methods and technical support for the diagnosis and differentiation of PSP.

[0006] To achieve the above object, the present invention provides the following solutions:

[0007] The present invention provides a biomarker for assisting in the diagnosis of progressive supranuclear palsy, wherein the biomarker is astrocyte-derived extracellular vesicles carrying Tau protein;

[0008] The Tau protein includes Tau or pTau181.

[0009] Furthermore, a higher concentration of the astrocyte-derived extracellular vesicles carrying Tau protein in plasma indicates a higher risk of progressive supranuclear palsy.

[0010] The present invention also provides use of a product for detecting the concentration of the above-mentioned biomarker in preparing a product for diagnosing progressive supranuclear palsy.

[0011] The present invention also provides a biomarker for distinguishing progressive supranuclear palsy and Parkinson's disease, wherein the biomarker is astrocyte-derived extracellular vesicles carrying Tau protein; the Tau protein includes Tau or pTau181.

[0012] Furthermore, the concentration of astrocyte-derived extracellular vesicles carrying Tau protein in the plasma of patients with progressive supranuclear palsy is higher than that in patients with Parkinson's disease.

[0013] The present invention also provides use of a product for detecting the concentration of the above-mentioned biomarker in the preparation of a product for identifying progressive supranuclear palsy and Parkinson's disease.

[0014] The present invention also provides a diagnostic model for progressive supranuclear palsy, wherein the input variable of the diagnostic model is the concentration of astrocyte-derived extracellular vesicles carrying Tau protein; the Tau protein includes Tau or pTau181;

[0015] A higher concentration of the astrocyte-derived extracellular vesicles carrying Tau protein in plasma indicates a higher risk of progressive supranuclear palsy.

[0016] The present invention also provides a diagnostic model for distinguishing progressive supranuclear palsy from Parkinson's disease, wherein the input variable of the diagnostic model is the concentration of astrocyte-derived extracellular vesicles carrying Tau protein; the Tau protein includes Tau or pTau181;

[0017] The concentration of the astrocyte-derived extracellular vesicles carrying Tau protein in the plasma of patients with progressive supranuclear palsy is higher than that in patients with Parkinson's disease.

[0018] Furthermore, the method for determining the concentration of astrocyte-derived extracellular vesicles carrying Tau protein is:

[0019] (1) Collect plasma samples;

[0020] (2) preparing fluorescently labeled antibodies against glutamate transporter 1 and Tau;

[0021] (3) mixing the plasma sample with the anti-glutamate transporter 1 fluorescently labeled antibody and incubating the mixture, then adding the Tau fluorescently labeled antibody and continuing the incubation to obtain a mixture;

[0022] (4) Using a nanoscale flow cytometer, the concentration of astrocyte-derived extracellular vesicles carrying Tau protein in the mixture is detected.

[0023] The present invention discloses the following technical effects:

[0024] The present invention uses fluorescently labeled specific Tau protein antibodies and anti-glutamate transporter 1 (Anti-Glutamate Transporter 1) antibodies to accurately detect astrocyte-derived extracellular vesicles (EVs) carrying Tau in plasma. By analyzing the concentration of astrocyte-derived EVs carrying Tau in plasma, it can accurately distinguish PSP patients from PD patients and healthy people, providing an efficient PSP diagnostic biomarker and diagnostic model.

[0025] In the process of constructing a diagnostic model, the present invention significantly enhances the ability to detect various forms of Tau protein and specific cells through the combination of different fluorescently labeled antibodies, providing detection sensitivity and specificity. Flow cytometry is used to quantitatively analyze vesicles with a diameter of less than 500 nm. The high-throughput characteristics of flow cytometry enable rapid acquisition of large amounts of data in a single test, thereby improving analysis efficiency and consistency of results. The entire detection process can be completed within 4 hours, maintaining sample freshness and ensuring timely detection. By combining fluorescent labeling of specific antibodies with an efficient sample processing process, the present invention greatly enhances the ability to diagnose PSP early, effectively solving the problems of complexity and low sensitivity faced by traditional diagnostic methods. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0027] Figure 1The characteristic analysis results of EVs; A is the TEM image of EVs; B is the Western blot analysis of EVs obtained by ultracentrifugation; C is the distribution of nanoparticles in EVs of the PSP group; D is a comparison of the concentrations of GLT-1-positive EVs in plasma (plasma GLT-1), GLT-1-positive EVs in ultracentrifuged plasma supernatant (UC supernatant GLT-1), and IgG-positive EVs in plasma (plasma IgG) in the PSP group;

[0028] Figure 2 is the concentration of astrocyte-derived EVs carrying pTau181 in the plasma of PSPs, PDs, and HCs;

[0029] Figure 3 ROC curve of astrocyte-derived EVs carrying pTau181 in plasma for differentiating PSP from PD;

[0030] Figure 4 ROC curve of astrocyte-derived EVs carrying pTau181 in plasma for distinguishing PSP from HC. DETAILED DESCRIPTION

[0031] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0032] It should be understood that the terms described herein are intended only to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges herein, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. The intermediate value within any stated value or stated range, and each smaller range between any other stated value or intermediate value within the stated range, is also encompassed within the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0033] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of any conflict with any incorporated document, the contents of this specification shall prevail.

[0034] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the description of the invention. The description and examples are intended to be illustrative only.

[0035] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0036] Example 1

[0037] 1. Experimental Materials

[0038] Included samples: 105 patients with progressive supranuclear palsy (PSP), 80 patients with Parkinson's disease (PD), and 80 healthy controls (HC).

[0039] Plasma Sample Preparation: Venous blood samples were collected from participants in the morning, fasting, using tubes coated with ethylenediaminetetraacetic acid. Blood samples were centrifuged at 1500 × g for 15 minutes at 4°C, and the supernatant was centrifuged at 12,000 × g for 30 minutes at 4°C. The supernatant plasma was stored at −80°C for use in nanoscale flow cytometry and nanoparticle tracking analysis (NTA).

[0040] Isolation of astrocyte-derived EVs: 10 μg of Anti-Glutamate Transporter 1 (Anti-GLT-1) antibody was coated onto 1 mg of M-270 epoxy beads using the Dynabeads Antibody Coupling Kit (14311D, Invitrogen) according to the manufacturer's instructions. Anti-Glutamate Transporter 1-labeled EVs were isolated from plasma EVs using immunocapture technology.

[0041] 2. Experimental Methods

[0042] Transmission electron microscopy (TEM) analysis: 5 μL of Anti-Glutamate Transporter 1-labeled EVs were placed on a copper grid coated with a carbon support film and incubated for 60 seconds. Excess sample was removed with filter paper. The copper grid was then stained with a 2% uranyl acetate solution for 60 seconds. Excess uranyl acetate was removed with filter paper, and the sample was air-dried. Electron microscopy images were acquired using a Tecnai F20 transmission electron microscope at 200 kV.

[0043] Western blot analysis: The total protein concentration of EVs obtained by ultracentrifugation was assessed using the BCA assay. 15 μg of total protein from the samples was separated on a 4-20% Bis-Tris gel (M42010C, Genscript) and then electrotransferred to a nitrocellulose membrane. After blocking with 5% skim milk, the membrane was incubated overnight at 4°C with anti-glutamate transporter 1 antibody, AliX polyclonal antibody, TSG 101 polyclonal antibody, Actin polyclonal antibody, and CD9 monoclonal antibody.

[0044] Nanoparticle Tracking Analysis: 2 μL plasma samples from PSP patients were diluted 1:500 in PBS (pH 7.4). These diluted samples were then analyzed using the ZetaView platform (Particle Metrix) to assess the distribution and concentration of nanoparticles.

[0045] EV analysis using a Cytoflex nanoscale flow cytometer: Antibodies were conjugated to fluorophores using the Zenon IgG labeling kit according to the kit's instructions. Anti-Glutamate Transporter 1 (astrocyte-derived) and pTau181 antibodies were labeled using different fluorescent labeling kits. 5 μL of plasma was placed in a flow cytometer tube. 0.1 μg of fluorescently conjugated anti-Glutamate Transporter 1 antibody was then added and incubated for 30 minutes, followed by 0.2 μg of fluorescently conjugated pTau181 antibody and incubated for 20 minutes. All procedures were performed at room temperature in the dark. After incubation, the mixture was diluted 1:60 with PBS, vortexed, and centrifuged for 10 seconds. Vesicles smaller than 500 nm were quantified using a nanoscale flow cytometer on a Cytoflex S platform (Beckman Coulter, Milano, Italy) in VSSC-H mode. The concentration of EVs carrying pTau181 protein and Anti-Glutamate Transporter 1 was calculated based on the flow rate and PBS dilution ratio. All samples were tested within 4 hours and analyzed by single-batch cell counting on the same day.

[0046] 3. Data Processing

[0047] Statistical analyses were performed using GraphPad Prism 10 and SPSS 26.0. Intergroup differences in the mean concentration of tau-carrying astrocyte-derived EVs were assessed using the Kruskal-Wallis test, followed by post hoc comparisons between PSP and PD, and between PSP and HC, using the Dunn test. Receiver operating characteristic (ROC) curve analysis was used to assess the sensitivity and specificity of these markers in distinguishing PSP from HCs or PD. Spearman correlation analysis was used to assess the correlation between clinical indicators and the concentration of tau-carrying astrocyte-derived EVs in the PSP group, with the Bonferroni correction adjusted for multiple comparisons. All tests were two-tailed, and statistical significance was considered to be P < 0.05.

[0048] 4. Results and Analysis

[0049] 1. TEM analysis results:

[0050] TEM analysis showed that the diameter of astrocyte-derived EVs was approximately 100 nm ( Figure 1 A).

[0051] 2. Western blot analysis results:

[0052] Western blot analysis showed that GLT-1 was enriched in reference EVs samples obtained by ultracentrifugation, along with the general EV markers Alix, TSG 101, Actin, and CD9 ( Figure 1 B).

[0053] 3. Nanoparticle tracking analysis results:

[0054] Nanoparticle tracking analysis showed that GLT-1-positive EVs in PSP plasma showed a broad peak centered at 100 nm ( Figure 1 The concentrations of GLT-1-positive EVs in PSP plasma, GLT-1-positive EVs in ultracentrifuged plasma supernatant, and IgG-positive EVs in PSP plasma (for control) were as shown in Figure 5C. Figure 1 As shown in D.

[0055] 4. Nanoscale flow cytometer analysis results:

[0056] like Figure 2As shown, astrocyte-derived EVs carrying pTau181 can effectively distinguish PSP from PD and HC: among the 105 PSP patients, 80 PD patients and 80 HC included, the concentration of astrocyte-derived EVs containing pTau181 in the plasma of PSP patients was significantly higher than that of HC and PD patients (PSP vs HCs: PpTau181 < 0.0001; PSP vs PD: PpTau181 < 0.0001; Figure 2 ).

[0057] ROC analysis evaluated the diagnostic utility of astrocyte-derived EVs carrying pTau181 in plasma in differentiating PSP from PD and HC. The area under the curve (AUC) value for differentiating PSP from PD was 0.815 (95% CI 0.752-0.877) ( Figure 3 ). In distinguishing PSP from HC, the AUC value was 0.917 (95% CI 0.876-0.958) ( Figure 4 When the concentration of astrocyte-derived EVs carrying pTau181 in plasma is less than 1,071,500 cells / mL, the patient is judged to have a low risk of Parkinson's disease or progressive supranuclear palsy; when the concentration of astrocyte-derived EVs carrying pTau181 in plasma is between 1,071,500 and 1,600,500 cells / mL, the patient is judged to have a high risk of Parkinson's disease and a low risk of progressive supranuclear palsy; when the concentration of astrocyte-derived EVs carrying pTau181 in plasma is greater than 1,600,500 cells / mL, the patient is judged to have a high risk of progressive supranuclear palsy.

[0058] The above results indicate that by evaluating the number or concentration of astrocyte-derived EVs carrying pTau181 in plasma, PSP patients and PD patients, as well as PSP patients and HC populations, can be effectively distinguished, thereby achieving the effect of auxiliary diagnosis of PSP.

[0059] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by persons skilled in the art should fall within the scope of protection defined by the claims of the present invention.

Claims

1. Use of a product for detecting the concentration of astrocyte-derived extracellular vesicles carrying Tau protein in the preparation of a product for diagnosing progressive supranuclear palsy, characterized in that: The Tau protein is pTau181; A higher concentration of the astrocyte-derived extracellular vesicles carrying Tau protein in plasma indicates a higher risk of progressive supranuclear palsy.

2. The use according to claim 1, characterized in that The method for determining the concentration of astrocyte-derived extracellular vesicles carrying Tau protein is as follows: (1) Collect plasma samples; (2) preparing fluorescently labeled antibodies against glutamate transporter 1 and pTau181; (3) mixing the plasma sample with the anti-glutamate transporter 1 fluorescently labeled antibody and incubating the mixture, then adding the pTau181 fluorescently labeled antibody and continuing the incubation to obtain a mixture; (4) Using a nanoscale flow cytometer, the concentration of astrocyte-derived extracellular vesicles carrying Tau protein in the mixture is detected.

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