Biomarkers for auxiliary diagnosis of progressive supranuclear palsy and their applications

Abstract

The present invention discloses a biomarker for assisting in the diagnosis of progressive supranuclear palsy and its application, and relates to the field of biomedical technology. The biomarker is a neuron-derived extracellular vesicle carrying a tau deformed protein; the tau deformed protein is 4R tau or pTau181. The present invention has developed a biomarker that can assist in the diagnosis of progressive supranuclear palsy, which can effectively distinguish patients with progressive supranuclear palsy (PSP), Parkinson's disease (PD) and healthy people (HC). At the same time, a diagnostic model for progressive supranuclear palsy was also constructed, wherein the multivariate logistic regression model had a sensitivity of 95.7% for distinguishing between PSP and HC, a specificity of 90.0%, and an AUC of 0.974; the multivariate logistic regression model had a sensitivity of 96.3%, a specificity of 90.0%, and an AUC of 0.968 for distinguishing between PSP and PD.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and in particular to biomarkers for auxiliary diagnosis of progressive supranuclear palsy and applications thereof. Background Art

[0002] Progressive supranuclear palsy (PSP) is a common atypical parkinsonian syndrome characterized by abnormal aggregation of tau protein in neurons, oligodendrocytes, and astrocytes. Diagnosis is complex due to the multiple clinical subtypes and symptom overlap between Parkinson's disease (PD) and other atypical parkinsonian syndromes. This diagnostic complexity often leads to a high misdiagnosis rate. Reliable biomarkers are urgently needed to facilitate early diagnosis and distinguish PSP from other neurodegenerative diseases.

[0003] Existing studies on tau protein detection have detected abnormal tau protein deposition in several biological fluids and peripheral tissues, including plasma, olfactory and oral epithelial cells, colon specimens, and skin biopsies. Compared to tissue biopsies, plasma is a routinely available clinical sample.

[0004] In the current study, disease-associated extracellular vesicles (EVs) were extracted and analyzed from plasma using methods such as immunocapture and enzyme-linked immunosorbent assay (ELISA), with a particular focus on Tau protein and its phosphorylated form. These methods require a large volume of serum sample (typically 500 μL), and the detection process is relatively complex and tedious.

[0005] While existing technologies have made some progress in biomarker research for neurodegenerative diseases, the following issues and drawbacks remain: High sample volume requirements: Existing technologies typically require serum samples of 500 μL or more; Complex detection procedures: Existing immunocapture and ELISA assays are cumbersome and time-consuming, requiring multiple steps, which can lead to errors between steps and affect the reliability and reproducibility of results; Inadequate sensitivity and specificity; and Lack of non-invasive detection methods: Existing biological samples must be obtained through invasive procedures such as tissue biopsies, making them difficult to widely use in conventional medical practice. The present invention aims to provide an effective and non-invasive detection method for the early diagnosis of PSP, thereby effectively distinguishing PSP from other neurodegenerative diseases. Summary of the Invention

[0006] The present invention aims to provide biomarkers and their applications for aiding the diagnosis of progressive supranuclear palsy (PSP) to address the aforementioned problems of the prior art. The present invention provides novel biomarkers and diagnostic models for aiding the diagnosis of PSP, particularly for differentiating PSP from Parkinson's disease, and has significant clinical application value.

[0007] To achieve the above object, the present invention provides the following solutions:

[0008] The present invention provides a biomarker for assisting in the diagnosis of progressive supranuclear palsy, wherein the biomarker is a neuron-derived extracellular vesicle carrying Tau deformable protein;

[0009] The Tau deformed protein is 4R Tau or pTau181.

[0010] Furthermore, a higher level of the biomarker in plasma indicates that the patient has a higher risk of developing progressive supranuclear palsy.

[0011] The present invention also provides a biomarker for distinguishing progressive supranuclear palsy and Parkinson's disease, wherein the biomarker is a neuron-derived extracellular vesicle carrying Tau deformable protein;

[0012] The Tau deformed protein is 4R Tau or pTau181.

[0013] Furthermore, a higher content of the biomarker in plasma indicates that the patient has a higher risk of developing progressive supranuclear palsy among progressive supranuclear palsy and Parkinson's disease.

[0014] The present invention also provides a diagnostic model for progressive supranuclear palsy, ND Tau / Tau, wherein the diagnostic model NDTau / Tau uses the plasma concentration of neuron-derived extracellular vesicles carrying Tau protein and the plasma concentration of total extracellular vesicles carrying Tau protein as input variables;

[0015] The ratio diagnostic model is risk value R=plasma concentration of neuron-derived extracellular vesicles carrying Tau protein / plasma concentration of total extracellular vesicles carrying Tau protein;

[0016] A higher value of the risk value R indicates that the patient has a higher risk of suffering from progressive supranuclear palsy.

[0017] The present invention also provides a diagnostic model ND pTau231 / pTau231 for progressive supranuclear palsy, wherein the diagnostic model ND pTau231 / pTau231 uses the plasma concentration of neuron-derived extracellular vesicles carrying the pTau231 protein and the plasma concentration of total extracellular vesicles carrying the pTau231 protein as input variables;

[0018] The ratio diagnostic model is risk value R=plasma concentration of neuron-derived extracellular vesicles carrying pTau231 protein / plasma concentration of total extracellular vesicles carrying pTau231 protein;

[0019] A higher value of the risk value R indicates that the patient has a higher risk of suffering from progressive supranuclear palsy.

[0020] The present invention also provides a diagnostic model ND 4R Tau / 4R Tau for distinguishing progressive supranuclear palsy from Parkinson's disease, wherein the diagnostic model ND 4R Tau / 4R Tau uses the plasma concentration of neuron-derived extracellular vesicles carrying 4R Tau protein and the plasma concentration of total extracellular vesicles carrying 4R Tau protein as input variables;

[0021] The ratio diagnostic model is: risk value R = plasma concentration of neuron-derived extracellular vesicles carrying 4R Tau protein / plasma concentration of total extracellular vesicles carrying 4R Tau protein;

[0022] A higher value of the risk value R indicates that the patient has a higher risk of suffering from progressive supranuclear palsy among progressive supranuclear palsy and Parkinson's disease.

[0023] The present invention also provides a multivariate logistic regression model for assisting in the diagnosis of progressive supranuclear palsy. When used to distinguish patients with progressive supranuclear palsy from normal people, the risk value D1 of the multivariate logistic regression model is D1 = 0.0000054431 × C tau +0.0000043461×C 4R Tau +0.0000004477×C pTau181 +0.0000032264×C pTau231 -0.0000001555×C pTau396 ;

[0024] When used to distinguish patients with progressive supranuclear palsy from patients with Parkinson's disease, the risk value of the multivariate logistic regression model was D2 = 0.0000070751 × C tau +0.0000028567×C 4RTau +0.0000006862×C pTau181 +0.0000013800×C pTau231 -0.0000004485×C pTau396 ;

[0025] The C tau 、The C 4RTau 、The C pTau181 、The C pTau231 and the C pTau396 Represents the concentration of neuronal-derived extracellular vesicles containing Tau, 4R Tau, pTau181, pTau231, or pTau396 in plasma, respectively.

[0026] The present invention also provides the use of a reagent for detecting the content of the above-mentioned biomarker in plasma in the preparation of a kit for auxiliary diagnosis of progressive supranuclear palsy.

[0027] Furthermore, the reagent is a reagent for nanoscale flow cytometry detection.

[0028] The present invention discloses the following technical effects:

[0029] The present invention has developed biomarkers that can assist in the diagnosis of progressive supranuclear palsy (PSP), effectively differentiating patients with progressive supranuclear palsy (PSP), Parkinson's disease (PD), and healthy controls (HC). A diagnostic model for PSP was also constructed. The multivariate logistic regression model achieved a sensitivity of 95.7%, a specificity of 90.0%, and an AUC of 0.974 for differentiating between PSP and HC; and a sensitivity of 96.3%, a specificity of 90.0%, and an AUC of 0.968 for differentiating between PSP and PD.

[0030] The present invention provides a new biomarker and diagnostic model for auxiliary diagnosis of progressive supranuclear palsy, especially for differentiating progressive supranuclear palsy from Parkinson's disease, and has important clinical application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] 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.

[0032] Figure 1 Characteristic analysis results of EVs; A is the TEM image of EVs; B is the Western blot analysis of ultracentrifugation supernatant (UCSupernatant), ultracentrifuged EVs and untreated reference plasma samples (Raw Plasma); C is the distribution of nanoparticles in EVs of the PSP group; D is the distribution of nanoparticles in EVs of the PD group; E is the distribution of nanoparticles in EVs of the HCs group; F is a statistical graph of the total concentration of EVs of all particle sizes in the plasma of the PSP, PD and HCs groups;

[0033] Figure 2The figures are the results of concentration detection and diagnostic value analysis of neuronal-derived EVs carrying Tau or its variant proteins in plasma samples of different groups; AE are statistical graphs of the concentrations of neuronal-derived EVs containing Tau, 4R Tau, pTau181, pTau231 and pTau396 in the plasma of PSP, PD and HCs, respectively; FJ are statistical graphs of the ratio of neuronal-derived EVs carrying Tau or its variant proteins to the total amount of EVs carrying Tau or its variant proteins in the plasma of PSP, PD and HC patients, respectively; K is the receiver operating characteristic curve of neuronal-derived EVs carrying Tau or its variant proteins in plasma for distinguishing PSP and HC patients; L is the receiver operating characteristic curve of the multivariate logistic regression model for distinguishing PSP and HC patients; M is the receiver operating characteristic curve of neuronal-derived EVs carrying Tau or its variant proteins in plasma for distinguishing PSP and PD patients; N is the receiver operating characteristic curve of the multivariate logistic regression model for distinguishing PSP and PD patients. DETAILED DESCRIPTION

[0034] 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.

[0035] 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.

[0036] 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.

[0037] 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 exemplary only.

[0038] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0039] Terminology Notes:

[0040] Tau protein is a microtubule-associated protein that is widely distributed in nerve cells of the nervous system and is an important component for stabilizing microtubules that serve as the skeleton of nerve cells.

[0041] 4R tau refers to Tau protein with four repeat regions.

[0042] pTau181, pTau231, and pTau396 are different phosphorylated forms of Tau protein, namely, phosphorylated Tau-181 protein, phosphorylated Tau-231 protein, and phosphorylated Tau-396 protein.

[0043] Example 1

[0044] 1. Materials and Methods

[0045] Included samples: 30 PSP patients, 27 PD patients and 23 healthy controls (HC).

[0046] Plasma Sample Preparation: Venous blood samples were collected from participants in the morning, fasting, using tubes coated with ethylenediaminetetraacetic acid. The blood samples were then centrifuged at 1500 × g for 15 minutes at 4°C to obtain the first supernatant. The first supernatant was then centrifuged at 12,000 × g for 30 minutes at 4°C to obtain the second supernatant, which served as the plasma sample. The second supernatant was stored at -80°C for use in nanoscale flow cytometry and nanoparticle tracking analysis (NTA).

[0047] Isolation of neuronal EVs: EVs carrying NMDAR2A were isolated from ultrapure plasma EVs using an immunocapture protocol. 10 μg of anti-NMDAR2A antibody was coated on 1 mg of M-270 epoxy beads using the Dynabeads Antibody Coupling Kit (14311D, Invitrogen) according to the manufacturer's instructions.

[0048] Transmission electron microscopy (TEM) analysis: 5 μL of NMRAR2A-labeled EVs was placed onto a copper grid coated with a carbon support film. After incubation 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.

[0049] Western blot analysis: Total protein concentrations in ultracentrifugation supernatants, ultracentrifuged EVs, and untreated reference plasma samples were assessed using the BCA assay. 15 μg of total protein from each sample was separated on a 4-20% Bis-Tris gel (M42010C, Genscript) and electrotransferred to a nitrocellulose membrane. After blocking with 5% nonfat milk, the membranes were incubated with anti-NMDAR2A antibodies, Ali polyclonal antibodies, and CD9 monoclonal antibodies overnight at 4°C.

[0050] Nanoparticle Tracking Analysis: 2 μL of plasma samples from PSP patients, PD patients, and HCs 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.

[0051] EVs were analyzed using a Cytoflex nanoscale flow cytometer: Antibodies were conjugated to fluorophores using the Zenon IgG labeling kit according to the manufacturer's instructions. Antibodies against NMDAR2A (to mark neuronal origin) and tau and its phosphorylated forms (e.g., pTau181, pTau231, and pTau396) were labeled using different fluorescent labeling kits. 5 μL of plasma was placed in a flow cytometer tube. 0.1 μg of fluorescently conjugated NMDAR2A antibody was then added and incubated for 30 minutes, followed by 0.2 μg of fluorescently conjugated tau antibody and incubation for 20 minutes, all 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 Tau and NMDAR2A was calculated based on the flow rate and PBS dilution ratio. All samples were assayed within 4 hours and analyzed in a single batch on the same day.

[0052] The present invention utilizes nanoscale flow cytometry for rapid and sensitive analysis of neuron-derived EVs in plasma. By combining fluorescent labeling of specific antibodies with an efficient sample processing process, it achieves the technical effect of improving the early diagnosis capability of progressive supranuclear palsy.

[0053] 2. Data Processing

[0054] Statistical analyses were performed using GraphPad Prism 10 and SPSS 26.0. The Kruskal-Wallis test was used to assess between-group differences in the mean concentration of tau-carrying neuronal EVs, followed by the Dunn test for comparisons between PSP and PD, and between PSP and HC. Receiver operating characteristic (ROC) curve analysis was used to assess the sensitivity and specificity of these markers in distinguishing PSP from HCs or PD. A multivariate model was constructed using binary logistic regression, incorporating various tau-carrying neuronal EVs, to distinguish PSP from PD or HC. Spearman correlation analysis was used to assess the correlation between clinical parameters and the concentration of tau-carrying neuronal 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.

[0055] 3. Results and Analysis

[0056] 1. Characteristics of EVs

[0057] TEM analysis showed that the diameter of neuronal EVs was approximately 100 nm ( Figure 1 Western blot analysis showed that NMDAR2A was enriched in reference EVs samples obtained by ultracentrifugation, along with the general EV markers Alix and CD9 ( Figure 1 NTA analysis showed that a broad peak centered at 100 nm appeared in the PD, PSP, and HC groups ( Figure 1 There was no significant difference in EV concentrations between these groups ( Figure 1 Middle F).

[0058] 2. Neuronal EVs carrying Tau can effectively distinguish PSP from PD and HC

[0059] Among the 30 PSP patients, 27 PD patients, and 23 HC patients included in the study, the concentrations of neuronal-derived EVs containing Tau, 4R Tau, and pTau181 in the plasma of PSP patients were significantly higher than those in HC and PD patients (PSP vs. HCs: P<0.05). Tau <0.0001, P 4RTau =0.004, P pTau181 <0.001; PSP compared with PD: P Tau <0.0001, P 4RTau <0.001, P pTau181 =0.005; Kruskal-Wallis test and Dunn's multiple comparison test, Figure 2 AC).

[0060] The present invention also analyzed the ratio of neuronal EVs carrying Tau, 4R Tau and pTau181, pTau231, and pTau396 to the total EVs carrying Tau (referred to as ratio). Figure 2 The ratio of neuronal EVs carrying Tau to EVs carrying Tau was significantly higher in PSP than in PD (P = 0.001) and HCs (P < 0.0001) ( Figure 2 In addition, the ratio of neuronal-derived EVs carrying 4RTau to the total number of EVs carrying 4R Tau was higher in PSP than in PD (P=0.003) ( Figure 2 Middle G), the ratio of neuronal-derived EVs carrying pTau231 to the total number of EVs carrying pTau231 was higher in PSPs than in HCs (P = 0.007) ( Figure 2 Middle I).

[0061] ROC analysis evaluated the diagnostic utility of neuronal EVs containing Tau, 4R Tau, pTau181, pTau231, and pTau396 in plasma for differentiating PSP from HC. The cut-off values ​​(unit: cells / mL) for these markers were as follows: Tau, 697,500; 4R Tau, 1,327,500; pTau181, 3,544,500. The area under the curve (AUC) values ​​for these markers were as follows: Tau, 0.938 (95% CI 0.877-0.998); 4R Tau, 0.765 (95% CI 0.636-0.893); pTau181, 0.800 (95% CI 0.681-0.919); pTau231, 0.644 (95% CI 0.495-0.792); pTau396, 0.634 (95% CI 0.481-0.787) ( Figure 2 Middle K).

[0062] In differentiating PSP from PD, the AUC values ​​of these neuronal EVs were Tau, 0.912 (95% CI 0.841-0.983); 4R Tau, 0.795 (95% CI 0.678-0.912); pTau181, 0.730 (95% CI 0.593-0.867); pTau231, 0.603 (95% CI 0.454-0.752); and pTau396, 0.519 (95% CI 0.366-0.673) ( Figure 2 The cut-off values ​​(unit: cells / mL) are as follows: Tau, 1,039,500; 4R Tau, 1,152,000; pTau181, 2,584,500.

[0063] 3. Multivariate logistic regression model analysis results

[0064] (1) Distinguishing between PSP and HC

[0065] The variables of the multivariate logistic regression model established in the present invention for distinguishing PSP from HC are shown in Table 1, and the risk value calculation equation is as follows:

[0066] Risk value D1 = 0.0000054431 × C tau +0.0000043461×C 4RTau +0.0000004477×C pTau181 +0.0000032264×C pTau231 -0.0000001555×C pTau396 ;

[0067] Where C tau 、C 4R Tau 、C pTau181 、C pTau231 and C pTau396 Represents the concentration of neuronal-derived EVs containing Tau, 4R Tau, pTau181, pTau231, or pTau396 in plasma, respectively.

[0068] Table 1 Equation variables of the multivariate logistic regression model for distinguishing PSP from HC

[0069] markers B Standard error Wald degrees of freedom Significance Exp(B) Tau 0.0000054431 0.0000020812 6.840 1 0.0089118534 1.0000054431 4Rtau 0.0000043461 0.0000028399 2.342 1 0.1259263804 1.0000043461 pTau181 0.0000004477 0.0000004736 0.894 1 0.3445216510 1.0000004477 pTau231 0.0000032264 0.0000028035 1.324 1 0.2497914587 1.0000032264 pTau396 -0.0000001555 0.0000003090 0.253 1 0.6149223604 0.9999998445 constant -14.9399425709 6.0845472380 6.029 1 0.0140731422 0.0000003248

[0070] The multivariate logistic regression model incorporating these neuronal EVs carrying Tau had an AUC of 0.974 (95% CI 0.941-1.000) and a cut-off value (unit: cells / mL) of 0.6267, indicating a sensitivity of 95.7% and a specificity of 90.0% for distinguishing PSP from HC ( Figure 2 Middle L).

[0071] (2) Distinguishing between PSP and PD

[0072] The variables of the multivariate logistic regression model established in the present invention for distinguishing PSP from PD are shown in Table 2, and the risk value calculation equation is as follows:

[0073] Risk value D2 = 0.0000070751 × C tau +0.0000028567×C 4RTau +0.0000006862×C pTau181 +0.0000013800×C pTau231 -0.0000004485×CpTau396 ;

[0074] Where C tau 、C 4R Tau 、C pTau181 、C pTau231 and C pTau396 Represents the concentration of neuronal-derived EVs containing Tau, 4R Tau, pTau181, pTau231, or pTau396 in plasma, respectively.

[0075] Table 2 Equation variables of the multivariable logistic regression model for distinguishing PSP from PD

[0076]

[0077]

[0078] The corresponding multivariate logistic regression model integrating these biomarkers had an AUC of 0.968 (95% CI 0.929-1.000), a cut-off value (unit: cells / mL) of 0.6398, a sensitivity of 96.3% and a specificity of 90.0% ( Figure 2 (in N).

[0079] 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. A method for constructing a multivariate logistic regression model for assisting the diagnosis of progressive supranuclear palsy, characterized in that: The multivariate logistic regression model was constructed using the concentrations of neuronal-derived extracellular vesicles containing Tau, 4R Tau, pTau181, pTau231, and pTau396 in plasma as input variables; When used to distinguish patients with progressive supranuclear palsy from normal subjects, the risk value of the multivariate logistic regression model is D1 = 0.0000054431 × C tau +0.0000043461×C 4R Tau +0.0000004477×C pTau181 +0.0000032264×C pTau231 -0.0000001555×C pTau396 ; When used to distinguish patients with progressive supranuclear palsy from patients with Parkinson's disease, the risk value of the multivariable logistic regression model was D2 = 0.0000070751 × C tau +0.0000028567×C 4R Tau +0.0000006862×C pTau181 +0.0000013800×C pTau231 -0.0000004485×C pTau396 ; The C tau 、The C 4R Tau 、The C pTau181 、The C pTau231 and the C pTau396 Represents the concentration of neuronal-derived extracellular vesicles containing Tau, 4R Tau, pTau181, pTau231, or pTau396 in plasma, respectively.

Images