Detection marker, diagnosis marker, application of detection marker and diagnosis marker, and kit

Abstract

The invention discloses a detection marker, a diagnosis marker, application of the detection marker and the diagnosis marker and a kit, and belongs to the technical field of medical medicines. The PPP2R5C is used as a detection marker for the preclinical stage of the Alzheimer's disease and as a diagnosis marker for mild cognitive impairment and the Alzheimer's disease, and the problems of detection defects of lumbar puncture and PET-CT detection methods used in AD early diagnosis and lack of effective blood biomarkers are solved.

Description

Detection markers, diagnostic markers, uses thereof, and kits Technical Field

[0001] The present invention relates to the field of medicine, and in particular to detection markers for the preclinical stage of Alzheimer's disease, diagnostic markers for AD-derived mild cognitive impairment, diagnostic markers for Alzheimer's disease and their uses, as well as corresponding kits; it also relates to a method for drug screening using the PPP2R5C protein, a composition comprising a reagent capable of detecting the PPP2R5C protein, and a marker composition containing the PPP2R5C protein. Background Art

[0002] Alzheimer's disease (AD) is a continuous disease spectrum, including mild, moderate and severe AD stages. In addition, before developing into AD, there are also preclinical stages of AD (pre-AD) and mild cognitive impairment (MCI). Among them, the preclinical stage has no clinical manifestations of cognitive impairment or only very mild memory loss, and may be related to the carrying of Alzheimer's disease-causing gene mutations. This stage can last for more than 20 years. It takes an average of only 2-6 years from MCI to mild AD, and 8-10 years from mild to severe AD.

[0003] Timely intervention can significantly slow the progression of Alzheimer's disease, but seizing the golden therapeutic window requires effective screening and diagnostic methods. Over the past four decades, thanks to the tireless efforts of researchers, AD diagnosis has made significant progress. In 2018, the National Institute on Aging and the Alzheimer's Association (NIA-AA) proposed the AT(N) diagnostic framework, based on the biological definitions of Aβ protein (A), hyperphosphorylated Tau protein (T), and neurodegeneration (N). This represents a significant breakthrough in recent years in using a combination of biomarkers to guide clinical AD diagnosis, allowing for more precise and early identification of the underlying disease type. However, the AT(N) framework shifts AD diagnosis from a clinical biological diagnosis to a purely biological definition separated from clinical phenotypes, generating controversy and challenges in routine clinical application. Currently, AD diagnosis still requires a combination of biomarkers based on clinical phenotypes. Furthermore, cerebrospinal fluid (CSF) testing is highly invasive, requiring patients to undergo a painful lumbar puncture. Positron emission tomography-computed tomograph (PET-CT) examinations are limited by regional economic development, resulting in a limited number of hospitals offering these services, high costs, and the presence of radiation. These factors limit the widespread clinical application of cerebrospinal fluid (CSF) testing and PET-CT, making them unsuitable for early screening and dynamic assessment.

[0004] Traditionally, AD is considered a brain-specific disease, and research on AD has primarily focused on pathological changes in the CNS. However, a growing body of research suggests that plasma proteins may also undergo AD-specific changes. Therefore, elucidating changes in plasma proteins may provide additional clues to understanding the pathogenesis of AD and aid in early screening and diagnosis. Compared to PET-CT and cerebrospinal fluid testing, blood testing has attracted considerable attention due to its minimally invasive, convenient, low-cost, and reproducible nature. Combined with other clinical information, it holds promise for the early identification and dynamic assessment of AD patients, as well as for risk assessment in healthy individuals.

[0005] Summary of the Invention

[0006] The present invention provides detection markers for the preclinical stage of Alzheimer's disease, diagnostic markers for mild cognitive impairment, AD diagnostic markers and their uses, as well as corresponding kits, aiming to address the detection defects currently existing in the early diagnosis of AD and fill the gap in detection methods in the pre-AD stage.

[0007] The inventors of the present invention used the method of mass spectrometry omics analysis to conduct a comprehensive analysis of neuronal-derived exosome proteins in the plasma of three groups of AD patients (AD group) who were positive for AT(N) biomarkers or had evidence of pathogenic gene mutations and a clear clinical diagnosis, and familial AD patients (pre-AD group) who carried AD pathogenic gene mutations and had a clear genetic diagnosis, and a healthy control group (CN) as the screening research subjects. They screened out the early diagnostic indicator PPP2R5C, and then used patients with sporadic AD and mild cognitive impairment (MCI) as the validation research subjects to determine the diagnostic efficacy of the candidate indicators. It was found that the level of PPP2R5C was gradually decreased in MCI and AD patients, showing a trend of healthy control>MCI>AD. Furthermore, by the ELISA method, the inventors directly tested the content of PPP2R5C in the plasma of the AD group, MCI group and healthy control group, and also showed a trend of healthy control>MCI>AD, thus proving that PPP2R5C in plasma can be used as a biomarker for the diagnosis of MCI and AD. Furthermore, regarding the mechanism by which PPP2R5C affects AD, the inventors discovered through research that PPP2R5C reduces both total and phosphorylated Tau in neurons, leading to a reduction in toxic neurofibrillary tangles (NFTs) in neurons, thereby protecting neuronal activity and reducing the risk of AD development and progression. Conversely, a decrease in PPP2R5C leads to an increase in total and phosphorylated Tau, an increase in toxic neurofibrillary tangles formed in neurons, leading to neuronal death and inducing the development of AD.

[0008] The PPP2R5C described in the present invention belongs to the phosphatase 2A (PP2A) regulatory subunit B family. It is also known as B56G, B56γ, and PR6G. PP2A is a major serine / threonine phosphatase that plays an important role in phosphorylation signal balance, affecting the phosphorylation state of many proteins and is associated with the regulation of several signal transduction pathways, cell proliferation and differentiation, cell malignant transformation, cell cycle regulation, DNA replication / transcription, and embryonic development. PP2A consists of three parts: a catalytic subunit (C), a structural subunit (B), and a regulatory subunit (A). Normally, the A and C subunits combine to form a heterodimer within the cell. AC is the core enzyme of PP2A, which can bind to the B subunit to form the heterotrimeric PP2A holoenzyme. Regulatory subunit B contains four different family members, including B (B55 / PR56), B' (B56 / PR56), B" (PR48) and B"' (PR93 / 110). The B' subunit has the greatest diversity, including α (PPP2R5A), β (PPP2R5B), γ (PPP2R5C), δ (PPP2R5D) and ε (PPP2R5E).

[0009] The PPP2R5C of the present invention can be one of multiple isomers of the PPP2R5C protein, or a combination of two or more of these isomers. In some embodiments, the amino acid sequence of the PPP2R5C is as shown in SEQ ID NO: 3. However, it should be understood that the PPP2R5C having this amino acid sequence is only an example of one of the isomers.

[0010] In a first aspect, the present invention provides a detection marker for the preclinical stage of Alzheimer's disease, wherein the detection marker is PPP2R5C protein.

[0011] In some embodiments, the subject in the preclinical stage of Alzheimer's disease carries an Alzheimer's disease-causing gene mutation, and the Alzheimer's disease-causing gene mutation includes at least one of a PSEN1 gene mutation, a PSEN2 gene mutation, and an APP gene mutation.

[0012] In a second aspect, the present invention provides use of PPP2R5C protein in preparing a preparation for detecting whether a subject is in the preclinical stage of Alzheimer's disease.

[0013] In some embodiments, the subject is in a preclinical stage of Alzheimer's disease and carries a mutation in a gene that causes Alzheimer's disease.

[0014] In some embodiments, the PPP2R5C protein is differentially expressed in the subject sample compared to the control sample.

[0015] In some embodiments, the expression of the PPP2R5C protein in the subject sample is reduced, and the subject is considered to be at least in the preclinical stage of Alzheimer's disease. Preferably, the expression of the PPP2R5C protein in the subject sample in the preclinical stage of Alzheimer's disease is lower than that in a control sample from a normal subject, and higher than that in a subject sample with mild cognitive impairment.

[0016] In some embodiments, the control sample and the subject sample are from plasma, brain tissue, or cerebrospinal fluid.

[0017] In some embodiments, the control sample and the subject sample comprise exosomes, wherein the exosomes comprise PPP2R5C protein. Preferably, the exosomes are derived from neurons in plasma.

[0018] In some embodiments, the subject is a mammal, preferably a human.

[0019] In a third aspect, the present invention provides a PPP2R5C protein for detecting whether a subject is in the preclinical stage of Alzheimer's disease.

[0020] In a fourth aspect, the present invention provides a method for detecting whether a subject is in a preclinical stage of Alzheimer's disease, comprising:

[0021] (i) detecting PPP2R5C protein in a sample from the subject using a reagent for detecting PPP2R5C protein; and

[0022] (ii) comparing the PPP2R5C protein with a control sample, wherein the difference in the PPP2R5C protein compared to the control sample is used to detect whether the subject is in a preclinical stage of Alzheimer's disease.

[0023] In some embodiments, the expression of the PPP2R5C protein in the sample from the subject is reduced, and the subject is considered to be in the preclinical stage of Alzheimer's disease.

[0024] In some embodiments, the control sample and the subject sample are from plasma, brain tissue, or cerebrospinal fluid.

[0025] In some embodiments, the control sample and the subject sample comprise exosomes, wherein the exosomes comprise PPP2R5C protein. Preferably, the exosomes are derived from neurons in plasma.

[0026] In some embodiments, the subject is a mammal, preferably a human.

[0027] In a fifth aspect, the present invention provides a kit for detecting whether a subject is in the preclinical stage of Alzheimer's disease, and the kit comprises a reagent for detecting the level of PPP2R5C.

[0028] In some embodiments, the PPP2R5C level is the protein level or mRNA level of PPP2R5C.

[0029] In some embodiments, the reagent detects the level of PPP2R5C by chromatography and / or mass spectrometry, fluorescence, electrophoresis, immunoaffinity, hybridization, immunochemistry, ultraviolet spectroscopy, fluorescence, radiochemical analysis, near infrared spectroscopy, nuclear magnetic resonance spectroscopy, light scattering analysis, and turbidimetry.

[0030] In some embodiments, the reagent for detecting the level of PPP2R5C comprises an antibody against the PPP2R5C protein. Preferably, the antibody is a monoclonal antibody or a polyclonal antibody; more preferably, a monoclonal antibody.

[0031] In some embodiments, the reagent for detecting the level of PPP2R5C comprises a reagent for detecting the level of mRNA encoding PPP2R5C, preferably comprising primers and / or probes capable of quantifying the level of mRNA encoding PPP2R5C. In some specific embodiments, the sequences of the primers used are shown in SEQ ID NO: 1-2.

[0032] In some embodiments, the reagent measures PPP2R5C levels by quantitative PCR.

[0033] In some embodiments, the kit further comprises a processing reagent for processing the biological sample.

[0034] In some embodiments, the subject is a mammal, more preferably a human.

[0035] In some embodiments, the kit is used to distinguish between a normal subject and a subject in a preclinical stage of Alzheimer's disease.

[0036] In a sixth aspect, the present invention provides a diagnostic marker for mild cognitive impairment, wherein the diagnostic marker is PPP2R5C protein.

[0037] In some embodiments, the mild cognitive impairment includes AD-induced mild cognitive impairment and non-AD-induced mild cognitive impairment.

[0038] In a seventh aspect, the present invention provides a use of PPP2R5C protein in preparing a preparation for diagnosing, assisting in the diagnosis, or detecting mild cognitive impairment in a subject, or for assessing the risk of a subject suffering from mild cognitive impairment.

[0039] In some embodiments, the PPP2R5C protein is differentially expressed in the subject sample compared to the control sample.

[0040] In some embodiments, expression of the PPP2R5C protein is reduced in the sample from the subject.

[0041] In some embodiments, the expression of the PPP2R5C protein is lower in patients with mild cognitive impairment than in subjects in the preclinical stage of Alzheimer's disease.

[0042] In some embodiments, the expression of the PPP2R5C protein is higher in patients with mild cognitive impairment than in subjects with Alzheimer's disease.

[0043] In some embodiments, the control sample and the subject sample are from plasma, brain tissue, or cerebrospinal fluid.

[0044] In some embodiments, the control sample and the subject sample comprise exosomes, wherein the exosomes comprise PPP2R5C protein. Preferably, the exosomes are derived from neurons in plasma.

[0045] In some embodiments, the subject is a mammal, preferably a human.

[0046] In an eighth aspect, the present invention provides a PPP2R5C protein for diagnosing, assisting in the diagnosis, or detecting mild cognitive impairment in a subject, or for assessing the risk of a subject suffering from mild cognitive impairment.

[0047] In a ninth aspect, the present invention provides a method for diagnosing, assisting in the diagnosis, or detecting mild cognitive impairment in a subject, or assessing the risk of mild cognitive impairment in a subject, comprising:

[0048] (i) detecting PPP2R5C protein in a sample from the subject using a reagent for detecting PPP2R5C protein; and

[0049] (ii) comparing the PPP2R5C protein with a control sample, wherein the difference in the PPP2R5C protein compared with the control sample is used to diagnose, assist in diagnosis, detect mild cognitive impairment in a subject, or assess the risk of mild cognitive impairment in a subject.

[0050] In some embodiments, expression of the PPP2R5C protein is reduced in the sample from the subject.

[0051] In some embodiments, the control sample and the subject sample are from plasma, brain tissue, or cerebrospinal fluid.

[0052] In some embodiments, the control sample and the subject sample comprise exosomes, wherein the exosomes comprise PPP2R5C protein. Preferably, the exosomes are derived from neurons in plasma.

[0053] In some embodiments, the subject is a mammal, preferably a human.

[0054] In a tenth aspect, the present invention provides a kit for diagnosing, assisting in the diagnosis, or detecting mild cognitive impairment in a subject, or assessing the risk of mild cognitive impairment in a subject, and the kit comprises a reagent for detecting the level of PPP2R5C.

[0055] In some embodiments, the PPP2R5C level is the protein level or mRNA level of PPP2R5C.

[0056] In some embodiments, the reagent detects the level of PPP2R5C by chromatography and / or mass spectrometry, fluorescence, electrophoresis, immunoaffinity, hybridization, immunochemistry, ultraviolet spectroscopy, fluorescence, radiochemical analysis, near infrared spectroscopy, nuclear magnetic resonance spectroscopy, light scattering analysis, and turbidimetry.

[0057] In some embodiments, the reagent for detecting the level of PPP2R5C comprises an antibody against the PPP2R5C protein. Preferably, the antibody is a monoclonal antibody or a polyclonal antibody; more preferably, a monoclonal antibody.

[0058] In some embodiments, the reagent for detecting the level of PPP2R5C comprises a reagent for detecting the level of mRNA encoding PPP2R5C, preferably comprising primers and / or probes capable of quantifying the level of mRNA encoding PPP2R5C. In some specific embodiments, the sequences of the primers used are shown in SEQ ID NO: 1-2.

[0059] In some embodiments, the reagent measures PPP2R5C levels by quantitative PCR.

[0060] In some embodiments, the kit further comprises a processing reagent for processing the biological sample.

[0061] In some embodiments, the subject is a mammal, more preferably a human.

[0062] In some embodiments, the kit is used to distinguish between normal subjects and subjects with mild cognitive impairment.

[0063] In an eleventh aspect, the present invention provides a diagnostic marker for Alzheimer's disease, wherein the diagnostic marker is PPP2R5C protein.

[0064] In some embodiments, the Alzheimer's disease includes familial AD and sporadic AD.

[0065] In some embodiments, the Alzheimer's disease includes mild AD, moderate AD, and severe AD.

[0066] In a twelfth aspect, the present invention provides the use of PPP2R5C protein in preparing a preparation for diagnosing, assisting in the diagnosis, detecting whether a subject has Alzheimer's disease, or for assessing the risk of a subject having Alzheimer's disease.

[0067] In some embodiments, the PPP2R5C protein is differentially expressed in the subject sample compared to the control sample.

[0068] In some embodiments, expression of the PPP2R5C protein is reduced in the sample from the subject.

[0069] In some embodiments, the expression of the PPP2R5C protein is lower in patients with Alzheimer's disease than in patients with mild cognitive impairment.

[0070] In some embodiments, the expression of the PPP2R5C protein is higher in patients with mild Alzheimer's disease than in patients with moderate Alzheimer's disease.

[0071] In some embodiments, the expression of the PPP2R5C protein is higher in patients with moderate Alzheimer's disease than in patients with severe Alzheimer's disease.

[0072] In some embodiments, the control sample and the subject sample are from plasma, brain tissue, or cerebrospinal fluid.

[0073] In some embodiments, the control sample and the subject sample comprise exosomes, wherein the exosomes comprise PPP2R5C protein. Preferably, the exosomes are derived from neurons in plasma.

[0074] In some embodiments, the subject is a mammal, preferably a human.

[0075] In some embodiments, the formulation is used for early diagnosis of Alzheimer's disease.

[0076] In a thirteenth aspect, the present invention provides a PPP2R5C protein for diagnosing, assisting in the diagnosis, or detecting whether a subject has Alzheimer's disease, or for assessing the risk of a subject having Alzheimer's disease.

[0077] In a fourteenth aspect, the present invention provides a method for diagnosing, assisting in the diagnosis, or detecting whether a subject has Alzheimer's disease, or assessing the risk of a subject having Alzheimer's disease, comprising:

[0078] (i) detecting PPP2R5C protein in a sample from the subject using a reagent for detecting PPP2R5C protein; and

[0079] (ii) comparing the PPP2R5C protein with a control sample, wherein the difference in the PPP2R5C protein compared to the control sample is used to diagnose, assist in diagnosis, detect whether a subject has Alzheimer's disease, or assess the risk of a subject having Alzheimer's disease.

[0080] In some embodiments, expression of the PPP2R5C protein is reduced in the sample from the subject.

[0081] In some embodiments, the control sample and the subject sample are from plasma, brain tissue, or cerebrospinal fluid.

[0082] In some embodiments, the control sample and the subject sample comprise exosomes, wherein the exosomes comprise PPP2R5C protein. Preferably, the exosomes are derived from neurons in plasma.

[0083] In some embodiments, the subject is a mammal, preferably a human.

[0084] In some embodiments, the method is used for early diagnosis of Alzheimer's disease.

[0085] In a fifteenth aspect, the present invention provides a kit for diagnosing, assisting in the diagnosis, or detecting whether a subject has Alzheimer's disease, or assessing the risk of a subject having Alzheimer's disease, and the kit comprises a reagent for detecting the level of PPP2R5C.

[0086] In some embodiments, the PPP2R5C level is the protein level or mRNA level of PPP2R5C.

[0087] In some embodiments, the reagent detects the level of PPP2R5C by chromatography and / or mass spectrometry, fluorescence, electrophoresis, immunoaffinity, hybridization, immunochemistry, ultraviolet spectroscopy, fluorescence, radiochemical analysis, near infrared spectroscopy, nuclear magnetic resonance spectroscopy, light scattering analysis, and turbidimetry.

[0088] In some embodiments, the reagent for detecting the level of PPP2R5C comprises an antibody against the PPP2R5C protein. Preferably, the antibody is a monoclonal antibody or a polyclonal antibody; more preferably, a monoclonal antibody.

[0089] In some embodiments, the reagent for detecting the level of PPP2R5C comprises a reagent for detecting the level of mRNA encoding PPP2R5C, preferably comprising primers and / or probes capable of quantifying the level of mRNA encoding PPP2R5C. In some specific embodiments, the sequences of the primers used are shown in SEQ ID NO: 1-2.

[0090] In some embodiments, the reagent measures PPP2R5C levels by quantitative PCR.

[0091] In some embodiments, the kit further comprises a processing reagent for processing the biological sample.

[0092] In some embodiments, the subject is a mammal, more preferably a human.

[0093] In some embodiments, the kit is used to distinguish between a normal subject and a subject suffering from Alzheimer's disease.

[0094] In some embodiments, the kit is used for early diagnosis of Alzheimer's disease.

[0095] In a sixteenth aspect, the present invention provides a drug screening method comprising:

[0096] (i) administering an effective amount of a drug to be screened to a subject, and detecting the level of PPP2R5C protein in a sample from the subject using a reagent for detecting PPP2R5C protein;

[0097] (ii) When the PPP2R5C protein level is increased compared with that before administration of the drug to be screened, the drug to be screened can be used to treat mild cognitive impairment and / or Alzheimer's disease.

[0098] In a seventeenth aspect, the present invention provides a composition for detecting whether a subject is in the preclinical stage of Alzheimer's disease, or for diagnosing, assisting in the diagnosis, detecting whether a subject has mild cognitive impairment, or for assessing the risk of a subject having mild cognitive impairment, or for diagnosing, assisting in the diagnosis, detecting whether a subject has Alzheimer's disease, or for assessing the risk of a subject having Alzheimer's disease, wherein the composition comprises a reagent capable of detecting PPP2R5C protein.

[0099] In some embodiments, the subject in the preclinical stage of Alzheimer's disease carries a mutation in a gene causing Alzheimer's disease.

[0100] In an eighteenth aspect, the present invention provides a marker composition for detecting whether a subject is in the preclinical stage of Alzheimer's disease, or for diagnosing, assisting in the diagnosis, detecting whether a subject has mild cognitive impairment, or for assessing the risk of a subject having mild cognitive impairment, or for diagnosing, assisting in the diagnosis, detecting whether a subject has Alzheimer's disease, or for assessing the risk of a subject having Alzheimer's disease, wherein the marker composition contains PPP2R5C protein.

[0101] In some embodiments, the subject in the preclinical stage of Alzheimer's disease carries a mutation in a gene causing Alzheimer's disease.

[0102] The present invention overcomes the detection defects of the lumbar puncture and PET-CT detection methods used in the early diagnosis of AD and the problem of the lack of effective blood biomarkers. The experimental results of the present invention show that the level of PPP2R5C in plasma is significantly downregulated in patients with mild cognitive impairment and Alzheimer's disease, showing a trend of normal cognitive function controls > mild cognitive impairment patients > Alzheimer's disease patients, thereby proving that PPP2R5C can be used as a marker for the diagnosis of mild cognitive impairment and Alzheimer's disease. In addition, the experimental results of the present invention also show that compared with normal cognitive function controls, PPP2R5C protein is downregulated in subjects carrying Alzheimer's disease pathogenic gene mutations, and therefore can be used as a detection marker for Alzheimer's disease pathogenic gene mutations. BRIEF DESCRIPTION OF THE DRAWINGS

[0103] FIG1 is a characterization diagram of neuronal-derived exosomes in plasma in Example 1 of the present invention;

[0104] FIG2 is a diagram showing the mass spectrometry screening results of PPP2R5C in Example 2 of the present invention;

[0105] FIG3 is a graph showing the efficacy validation of the neuronal-derived exosome PPP2R5C protein in plasma as an early diagnostic marker for AD in Example 3 of the present invention;

[0106] FIG4 shows the determination of PPP2R5C protein levels in plasma of three groups of samples in Example 4 of the present invention and the validation of its efficacy as an early diagnostic marker for AD;

[0107] FIG5 is a diagram showing the WB detection results of PPP2R5C protein in human brain tissue in Example 6 of the present invention;

[0108] FIG6 is a diagram showing the results of the study on the intrinsic mechanism of PPP2R5C as an AD marker in Example 7 of the present invention, wherein “OE-PPP2R5C” indicates overexpression of PPP2R5C. DETAILED DESCRIPTION

[0109] The following examples are intended to illustrate the present invention but are not intended to limit the scope of the present invention. Any modifications or substitutions to the methods, steps, or conditions of the present invention without departing from the spirit and essence of the present invention are intended to be within the scope of the present invention. The reagents, kits, and instruments used in the following examples are all commercially available. The methods used in the examples are consistent with conventional methods unless otherwise specified.

[0110] The technical solution of the present invention is further elaborated in detail below in conjunction with embodiments.

[0111] Example 1 Extraction and characterization of neuronal exosomes from plasma

[0112] This example uses a modified precipitation method to extract neuronal exosomes from plasma. The specific steps are as follows:

[0113] 1) Take 500 μL of plasma, thaw at 4°C, and add 5 μL of protease inhibitors;

[0114] 2) Use a plasma exosome extraction kit (System Biosciences, Cat#EXOQ5A) to add 5 μL of plasma exosome precipitation reagent (Thrombin Plasma Prep for Exosome precipitation), mix at room temperature for 5 minutes, and centrifuge at 10,000 rpm for 5 minutes;

[0115] 3) Remove the supernatant, add 10 μL of Pierce Streptavidin Agarose Resin and 50 μL of 3% BSA, mix well, and incubate at 4°C for 5 h;

[0116] 4) Centrifuge the suspension at 2500 g for 10 min at 4°C and remove the supernatant, avoiding the precipitate below.

[0117] 5) Add 126 μL of Exosome Precipitation Solution to the supernatant and let it stand for 1 hour at 4°C;

[0118] 6) Centrifuge the suspension at 1500 g for 30 min at 4°C and discard the supernatant;

[0119] 7) Add 1 mL of PBS to the pellet, centrifuge at 1500 g for 5 min at 4°C, and discard the supernatant.

[0120] 8) Add 250 μL PBS, 5 μL protease inhibitors, 50 μL 3% BSA, 4 μL human CD171 antibody-linked biotin, and 25 μL Pierce streptavidin agarose resin to the pellet and incubate overnight at 4°C.

[0121] 9) Centrifuge the suspension at 200 g for 10 min at 4°C and discard the supernatant;

[0122] 10) Add 1.5 mL of PBS to the pellet, centrifuge at 400 g for 3 min at 4°C, and discard the supernatant.

[0123] 11) For samples undergoing transmission electron microscopy, add 50 μL of glycine buffer (pH 3.0) to the pellet and incubate at 4°C for 10 min. Place 10 μL of the exosome suspension onto a copper mesh grid for an electron microscope and allow it to remain on the grid for at least 30 min. Fix with 50 μL of PBS for 1 min, repeat 3-4 times. Fix with 10 μL of 4% glutaraldehyde for 5 min, blot dry with filter paper, wash once with PBS, and air-dry at room temperature. Observe and photograph the washed copper grid under a Hitachi-7500 transmission electron microscope.

[0124] 12) Determine the particle size of exosomes;

[0125] 13) Neuronal exosomes were characterized by immunoblotting using the neuronal exosome-specific antibody CD171 (Abcam, #270455) and the exosome-specific antibody Alix (Abcam, #275377).

[0126] The electron microscopic characterization of the morphology of neuronal-derived exosomes in plasma in this example is shown in Figure 1a, the exosome particle size distribution is shown in Figure 1b, and the specific characterization of exosomes belonging to neuronal origin is shown in Figure 1c.

[0127] As shown in Figure 1a, the neuronal exosomes isolated from plasma have typical morphology and are not ruptured.

[0128] As shown in Figure 1b, the particle size distribution of neuronal exosomes isolated from plasma is uniform, with an average particle size of 107.3 nm.

[0129] As shown in Figure 1c, the exosomes isolated from plasma are secreted by neurons.

[0130] Example 2 Screening of PPP2R5C protein

[0131] This example included 41 subjects, including 13 genetically diagnosed FAD (familial AD), 10 asymptomatic carriers of AD pathogenic gene mutations (pre-AD), and 18 normal controls (CN). To reduce intra-group variability, these samples were pooled. Based on the different gene mutations, the 13 FAD patients were pooled into five groups: two each with PSEN1 and PSEN2 mutations, one with APP mutations, and three with age- and sex-matched normal controls. Similarly, the pre-AD patients were divided into five groups: three with PSEN1 mutations, one with PSEN2 and one with APP mutations, and three with age- and sex-matched normal controls. Plasma neuronal exosomes were extracted from the 41 subjects, and their proteomes were qualitatively, quantitatively, and subjected to bioinformatics cluster heat map analysis using label-free LC-MS / MS. Differentially expressed proteins shared by the FAD and pre-AD groups were identified, with the expression level of PPP2R5C protein found to be higher in CN than in pre-AD and higher in AD.

[0132] The label-free LC-MS / MS technology used for the neuronal exosomes mentioned above is as follows:

[0133] 10 μg of exosome protein samples obtained by the modified precipitation method were subjected to 1-hour gradient Q-exactive analysis, and a total of 3 LC-MS / MS analyses, which mainly included filter-assisted sample preparation, nanoliter flow rate high-performance liquid chromatography liquid phase system Easy nLC separation, and all samples were separated by high-performance liquid chromatography. After protein mass spectrometry analysis was performed on the Q-Exactive mass spectrometer, the mass spectrometry detection time was 2 hours, positive ion detection was used as the detection mode, the scan range was set to 300 to 1800 m / z for the parent ion, and the resolution of the primary mass spectrometer was required to reach 70,000 at 200 m / z, and the automatic control target was set to 1e 6 The maximum IT was 50 milliseconds and the dynamic exclusion time was 60.0 seconds. MaxQuant software (version 1.5.3.17) was used for library search and identification.

[0134] The results of the differential protein clustering heat map analysis of the mass spectrometry results in this example are shown in Figure 2a, and the relative abundance statistical graph of the PPP2R5C protein is shown in Figure 2b.

[0135] As shown in Figure 2a, PPP2R5C (protein ID H0YN58) showed the most significant changes in the healthy control group (CN), pre-AD group, and AD group, with a gradually decreasing trend. Cluster C represents CN, PER represents pre-AD, and P represents patients with AD.

[0136] As shown in Figure 2b, the abundance of PPP2R5C measured by mass spectrometry was decreased in both the pre-AD group and the AD group compared with the healthy control, and the decrease was more obvious in the AD group, with statistical differences between the two groups.

[0137] Example 3: Validation of the efficacy of neuronal exosomal PPP2R5C protein in plasma as an early diagnostic marker for AD

[0138] A total of 64 research subjects were included in this example, including 20 AD patients, 12 mild cognitive impairment (MCI) patients (including 4 patients with AD-derived mild cognitive impairment and 8 patients with non-AD-derived mild cognitive impairment), and 32 age- and sex-matched normal controls. The modified precipitation method was used to extract plasma neuron-derived exosome proteins from all research subjects, and the parallel reaction monitoring (PRM) method was used to quantitatively detect PPP2R5C. The expression levels of candidate proteins were compared between mild AD / normal control, MCI / normal control, and mild AD / MCI, and differentially expressed proteins were screened and obtained. The subject characteristic curves were constructed as shown in Figures 3a to 3c. The diagnostic efficacy of different indicators for mild AD and MCI was compared, and it was found that PPP2R5C in plasma neuron-derived exosomes may be used as an indicator for early diagnosis of AD and may change before symptoms appear.

[0139] The validation criteria were selected based on the following criteria for candidate early diagnostic biomarkers: 1. Differentially expressed proteins shared by FAD and pre-AD, and / or proteins associated with dementia; 2. Meet the technical requirements for PRM testing, i.e., each protein must have two or more unique peptides identified. The differentially expressed proteins and dementia-associated proteins in Example 2 were screened, and a total of 26 proteins met the selection criteria for further validation in a sporadic population.

[0140] The PRM method involves the following steps: During the PRM method development phase, four samples were randomly selected from each group, each containing 20 μg of protein. Proteolysis was performed using a LysC+Trypsin protocol. The hydrolysis products were desalted and lyophilized, and the peptides were resolubilized in 0.1% FA. Peptide concentrations were determined by OD280 for mass spectrometry analysis. An appropriate amount of hydrolyzed peptides was collected from each group, and the 12 samples were pooled within each group to create three mixed samples. Based on the sequences of 26 target proteins and the results of the initial screening, specific peptides for the target proteins were initially selected for subsequent PRM analysis. LC-MS / MS analysis was performed on each of the three mixed samples, with a total mass spectrometry analysis time of 2 hours (2 x 2 hr). The mass spectrometry data were searched using Maxquant 1.5.3.17 and imported into Skyline software to identify credible peptides representing the target proteins and identify proteins suitable for PRM quantification. PRM method parameters were optimized to finalize the LC-PRM / MS method. Appropriate amounts of peptides were spiked into four mixed samples with an appropriate amount of AQUA internal standard peptide. LC-PRM / MS mass spectrometry was performed for 1 hour on each sample, with three replicates. Data were analyzed using Skyline, and peptides and product ions were selected for protein quantification.

[0141] In this embodiment, the ROC curve of PPP2R5C for distinguishing normal controls from MCI patients is shown in FIG3a , the ROC curve of PPP2R5C for distinguishing normal controls from AD patients is shown in FIG3b , and the ROC curve of PPP2R5C for distinguishing MCI patients from AD patients is shown in FIG3c .

[0142] As can be seen from Figures 3a-c, PPP2R5C in neuronal-derived exosomes in plasma has good diagnostic efficacy for distinguishing normal controls, MCI and AD, suggesting that PPP2R5C is an early screening marker for AD.

[0143] Example 4 Determination of PPP2R5C Protein Content in Plasma and Verification of Its Efficacy as an Early Diagnostic Marker for AD

[0144] A total of 226 subjects were included in this example, including 76 AD patients (AD), 76 mild cognitive impairment (MCI) patients (including 26 AD-derived MCI patients and 50 non-AD-derived MCI patients), and 74 age- and sex-matched normal controls (CN). The enzyme-linked immunosorbent assay (ELISA) was used to directly detect the level of PPP2R5C in plasma. The ELISA kit used was produced by Abbexa, USA, with the catalog number abx500105.

[0145] The ELISA assay results in this example are shown in FIG4a , and the efficacy verification analysis results are shown in FIG4b .

[0146] As shown in Figure 4a, compared with the normal control group, the plasma PPP2R5C protein content decreased in both the MCI group and the AD group, and the decrease was more obvious in the AD group. There was statistical significance between the two groups, CN>MCI>AD, which was consistent with the changes in PPP2R5C in neuronal-derived exosomes isolated from plasma.

[0147] As shown in Figure 4b, direct measurement of the plasma PPP2R5C content can distinguish normal controls from AD, and adding age and gender factors to the analysis can also effectively distinguish that the plasma PPP2R5C content is significantly reduced in AD.

[0148] This example demonstrates that PPP2R5C in plasma can be used as an early screening marker for AD.

[0149] Example 5 qRT-PCR detection of PPP2R5C mRNA levels in plasma

[0150] A total of 226 research subjects were included in this example, including 74 AD patients (AD), 76 mild cognitive impairment (MCI) patients (including 26 patients with AD-derived mild cognitive impairment and 50 patients with non-AD-derived mild cognitive impairment), and 76 age- and sex-matched normal controls (CN). Real-time fluorescence quantitative reverse transcription polymerase chain reaction (qRT-PCR) was used to detect the content of PPP2R5C mRNA in plasma. The reverse transcription kit used was produced by Themo Company with the product number K6225; the PCR kit was produced by QP Transgen with the product number AQ602-02. The primers used for PPP2R5C were:

[0151] h-PPP2R5C-F: 5′-CAGCGAAGATTCTGCCCATCATG-3′ (SEQ ID NO: 1)

[0152] h-PPP2R5C-R: 5′-TCATCTCCATGAAGAGCTTCAGG-3′ (SEQ ID NO: 2)

[0153] The qRT-PCR assay results in this example showed that compared with the normal control group (CN), the plasma PPP2R5C mRNA level decreased in both the MCI group and the AD group, and the decrease was more obvious in the AD group, showing a trend of CN>MCI>AD, which is consistent with the changes in the PPP2R5C protein level in neuronal exosomes isolated from plasma and the direct measurement of the PPP2R5C protein level in plasma.

[0154] This example demonstrates that the PPP2R5C mRNA level in plasma can be used as an early screening marker for AD.

[0155] Example 6 Detection of relative content of PPP2R5C protein in human brain tissue

[0156] This example included 19 subjects, including 7 AD patients, 7 elderly controls (>65 years old), and 5 young controls (20-40 years old). Western blotting was performed on brain tissue from each subject. The antibody against PPP2R5C used was a polyclonal antibody produced by Invitrogen, catalog number #PA5-110209.

[0157] The WB assay results in this example are shown in FIG5a , which indicate that PPP2R5C is significantly reduced in the brain tissue of AD patients. As shown in FIG5b , the statistical results show that both the elderly normal controls and the young normal controls are statistically different from the AD patients.

[0158] In the above examples, by using mass spectrometry analysis, familial AD patients and pre-AD patients with clear genetic diagnosis were used as screening research subjects, and their plasma neuron-derived exosome proteome was comprehensively analyzed to screen out PPP2R5C, a possible early diagnostic indicator for AD. Furthermore, AD and mild cognitive impairment (MCI) patients were used as validation research subjects to determine the diagnostic efficacy of the candidate indicators. It was found that PPP2R5C in plasma neuron-derived exosomes can be used as an early diagnostic marker for AD. At the same time, AD, mild cognitive impairment (MCI) patients and healthy controls were used as research subjects again, and ELISA and qRT-PCR detection methods were used to find that PPP2R5C protein in direct plasma can also be used as an early diagnostic marker for AD; and human brain tissue WB detection showed that PPP2R5C was also significantly reduced in AD patients.

[0159] Example 7 Study on the intrinsic mechanism of PPP2R5C as an AD marker

[0160] According to the previous literature search and the main physiological functions of PPP2R5C, this example uses AD transgenic mice Tau P301S to isolate and culture primary neurons in the hippocampus of fetal mice. When cultured for P12 days, adeno-associated viruses AAV-shPPP2R5C, AAV-PPP2R5C (Wuhan Shumi Brain Science Co., Ltd.) and corresponding control viruses are added to respectively knock down the expression of PPP2R5C in neurons by silencing or high expression. After 6 days of viral infection, neurons were collected and lysed, and immunoblotting (WB) was used to analyze the changes in total Tau and phosphorylated Tau in neurons after changes in PPP2R5C expression; changes in neuronal dendritic spines were also analyzed. The antibody information used are PPP2R5C antibody: Invitrogen / #PA5-110209, Tau5 antibody: Invitrogen / #MA5-12808, p-Tau T181 antibody: Invitrogen / #701530, p-Tau S396 antibody: Invitrogen / #44-752G, p-Tau S202 antibody: Invitrogen / #MN1020, GAPDH antibody: Prteintech / #1E6D9.

[0161] In this example, after PPP2R5C was overexpressed in primary hippocampal neurons of Tau P301S fetal rats, the WB detection results were shown in Figure 6a, the statistical graph of the WB results was shown in Figure 6b, and the neuronal dendritic spine staining results and statistical analysis were shown in Figure 6c and Figure 6d, respectively; after PPP2R5C was knocked down and silenced in primary hippocampal neurons of Tau P301S fetal rats, the WB detection results were shown in Figure 6e, the statistical graph of the WB results was shown in Figure 6f, and the neuronal dendritic spine staining results and statistical analysis were shown in Figure 6g and Figure 6h, respectively.

[0162] As shown in Figures 6a-d, after high expression of PPP2R5C in neurons, both total Tau and phosphorylated Tau decreased, and the dendritic spines of neurons increased significantly; after reducing PPP2R5C in neurons, both total Tau and phosphorylated Tau increased, and the dendritic spines of neurons decreased significantly.

[0163] Sequence information:

[0164] Amino acid sequence of PPP2R5C (SEQ ID NO: 3)

[0165] The above-described embodiments merely illustrate several implementations of the present invention, and while their descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that a person skilled in the art would be able to make numerous variations and improvements without departing from the spirit of the present invention, all of which fall within the scope of protection of the present invention. Therefore, the scope of protection of the present invention shall be determined by the appended claims.

Claims

1. Detection markers for preclinical stage of Alzheimer's disease, including: The detection marker is PPP2R5C protein.

2. The detection marker according to claim 1, wherein The subject in the preclinical stage of Alzheimer's disease carries a pathogenic gene mutation of Alzheimer's disease, and the pathogenic gene mutation of Alzheimer's disease includes at least one of a PSEN1 gene mutation, a PSEN2 gene mutation, and an APP gene mutation.

3. Use of PPP2R5C protein in the preparation of a preparation for detecting whether a subject is in the preclinical stage of Alzheimer's disease; preferably, the subject in the preclinical stage of Alzheimer's disease carries a mutation in a gene that causes Alzheimer's disease.

4. The use according to claim 3, wherein The PPP2R5C protein is differentially expressed in the subject samples compared to the control samples.

5. The use according to claim 3 or 4, wherein The expression of the PPP2R5C protein in the subject sample is reduced; Preferably, the expression of the PPP2R5C protein in samples from subjects at the preclinical stage of Alzheimer's disease is lower than that in control samples from normal subjects, and higher than that in samples from subjects with mild cognitive impairment.

6. The use according to any one of claims 3 to 5, wherein The control sample and the subject sample are from plasma, brain tissue or cerebrospinal fluid.

7. The use according to any one of claims 3 to 5, wherein The control sample and the subject sample contain exosomes, and the exosomes contain PPP2R5C protein; Preferably, the exosomes are derived from neurons in plasma.

8. The use according to any one of claims 3 to 7, wherein The subject is a mammal, preferably a human.

9. PPP2R5C protein used to detect whether a subject is in the preclinical stage of Alzheimer's disease.

10. A method for detecting whether a subject is in the preclinical stage of Alzheimer's disease, comprising: (i) detecting PPP2R5C protein in a sample from the subject using a reagent for detecting PPP2R5C protein; as well as (ii) comparing the PPP2R5C protein with a control sample, wherein the difference in the PPP2R5C protein compared to the control sample is used to detect whether the subject is in a preclinical stage of Alzheimer's disease; Preferably, the subject in the preclinical stage of Alzheimer's disease carries a mutation in a gene causing Alzheimer's disease.

11. The method according to claim 10, wherein: The expression of the PPP2R5C protein in the subject sample is reduced; Preferably, the expression of the PPP2R5C protein in the subject sample is lower than that in a control sample from a normal subject, and higher than that in a sample from a subject with mild cognitive impairment.

12. The method according to claim 10 or 11, wherein: The control sample and the subject sample are from plasma, brain tissue or cerebrospinal fluid.

13. The method according to claim 10 or 11, wherein: The control sample and the subject sample contain exosomes, and the exosomes contain PPP2R5C protein; Preferably, the exosomes are derived from neurons in plasma.

14. The method according to any one of claims 10 to 13, wherein: The subject is a mammal, preferably a human.

15. A kit, wherein: The kit is used to detect whether a subject is in the preclinical stage of Alzheimer's disease, and the kit comprises a reagent for detecting the level of PPP2R5C; Preferably, the subject in the preclinical stage of Alzheimer's disease carries a mutation in a gene causing Alzheimer's disease.

16. The kit according to claim 15, wherein The PPP2R5C level is the protein level or mRNA level of PPP2R5C; Preferably, the kit further comprises a processing reagent for processing the biological sample; Preferably, the subject is a mammal, more preferably a human.

17. The kit according to claim 15 or 16, wherein The reagent for detecting the level of PPP2R5C comprises an antibody against the PPP2R5C protein; Preferably, the antibody is a monoclonal antibody or a polyclonal antibody; more preferably a monoclonal antibody.

18. The kit according to claim 15 or 16, wherein The reagent for detecting the level of PPP2R5C comprises a reagent for detecting the level of mRNA encoding PPP2R5C, preferably comprising primers and / or probes capable of quantifying the level of mRNA encoding PPP2R5C; Preferably, the reagent is used to measure the level of PPP2R5C by quantitative PCR.

19. The kit according to any one of claims 15 to 18, wherein The kit is used to distinguish normal subjects from subjects carrying Alzheimer's disease-causing gene mutations.

20. Diagnostic markers for mild cognitive impairment, including: The diagnostic marker is PPP2R5C protein.

21. The diagnostic marker according to claim 20, wherein The mild cognitive impairment includes AD-induced mild cognitive impairment and non-AD-induced mild cognitive impairment.

22. Use of PPP2R5C protein in the preparation of a preparation for diagnosing, assisting in the diagnosis, detecting mild cognitive impairment in a subject, or assessing the risk of a subject suffering from mild cognitive impairment.

23. The use according to claim 22, wherein The PPP2R5C protein is differentially expressed in the subject samples compared to the control samples.

24. The use according to claim 22 or 23, wherein The expression of the PPP2R5C protein in the subject's sample is reduced.

25. The use according to any one of claims 22 to 24, wherein The expression of the PPP2R5C protein in patients with mild cognitive impairment is lower than that in subjects with Alzheimer's disease-causing gene mutations; and / or The expression of the PPP2R5C protein is higher in patients with mild cognitive impairment than in subjects with Alzheimer's disease.

26. The use according to any one of claims 22 to 25, wherein The control sample and the subject sample are from plasma, brain tissue or cerebrospinal fluid.

27. The use according to any one of claims 22 to 25, wherein The control sample and the subject sample contain exosomes, and the exosomes contain PPP2R5C protein; Preferably, the exosomes are derived from neurons in plasma.

28. The use according to any one of claims 22 to 27, wherein The subject is a mammal, more preferably a human.

29. A PPP2R5C protein used for diagnosing, assisting in the diagnosis, or detecting mild cognitive impairment in a subject, or for assessing the risk of a subject suffering from mild cognitive impairment.

30. A method for diagnosing, assisting in the diagnosis, or detecting a subject suffering from mild cognitive impairment, or assessing the risk of a subject suffering from mild cognitive impairment, comprising: (i) detecting PPP2R5C protein in a sample from the subject using a reagent for detecting PPP2R5C protein; as well as (ii) comparing the PPP2R5C protein with a control sample, wherein the difference in the PPP2R5C protein compared with the control sample is used to diagnose, assist in diagnosis, detect mild cognitive impairment in a subject, or assess the risk of mild cognitive impairment in a subject.

31. The method according to claim 30, wherein: The expression of the PPP2R5C protein in the subject's sample is reduced.

32. The method according to claim 30 or 31, wherein: The control sample and the subject sample are from plasma, brain tissue or cerebrospinal fluid.

33. The method according to any one of claims 30 to 31, wherein: The control sample and the subject sample contain exosomes, and the exosomes contain PPP2R5C protein; Preferably, the exosomes are derived from neurons in plasma.

34. The method according to any one of claims 30 to 33, wherein: The subject is a mammal, preferably a human.

35. A kit, wherein: The kit is used for diagnosing, assisting in diagnosis, detecting mild cognitive impairment in a subject, or assessing the risk of mild cognitive impairment in a subject, and the kit contains a reagent for detecting the level of PPP2R5C.

36. The kit according to claim 35, wherein The PPP2R5C level is the protein level or mRNA level of PPP2R5C; Preferably, the kit further comprises a processing reagent for processing the biological sample; Preferably, the subject is a mammal, more preferably a human.

37. The kit according to claim 35 or 36, wherein The reagent for detecting the level of PPP2R5C comprises an antibody against the PPP2R5C protein; Preferably, the antibody is a monoclonal antibody or a polyclonal antibody; more preferably a monoclonal antibody.

38. The kit according to claim 35 or 36, wherein The reagent for detecting the level of PPP2R5C comprises a reagent for detecting the level of mRNA encoding PPP2R5C, preferably comprising primers and / or probes capable of quantifying the level of mRNA encoding PPP2R5C; Preferably, the reagent is used to measure the level of PPP2R5C by quantitative PCR.

39. The kit according to any one of claims 35 to 38, wherein The kit is used to distinguish between normal subjects and subjects with mild cognitive impairment.

40. Alzheimer's disease diagnostic markers, including: The diagnostic marker is PPP2R5C protein.

41. The diagnostic marker according to claim 40, wherein The Alzheimer's disease includes familial AD and sporadic AD; and / or The Alzheimer's disease includes mild AD, moderate AD and severe AD.

42. Use of PPP2R5C protein in the preparation of a preparation for diagnosing, assisting in the diagnosis, detecting whether a subject has Alzheimer's disease, or for assessing the risk of a subject having Alzheimer's disease.

43. The use according to claim 42, wherein The PPP2R5C protein is differentially expressed in the subject samples compared to the control samples.

44. The use according to claim 42 or 43, wherein The expression of the PPP2R5C protein in the subject's sample is reduced.

45. The use according to any one of claims 42 to 44, wherein The expression of the PPP2R5C protein in patients with Alzheimer's disease is lower than that in patients with mild cognitive impairment.

46. ​​The use according to any one of claims 42 to 45, wherein The control sample and the subject sample are from plasma, brain tissue or cerebrospinal fluid.

47. The use according to any one of claims 42 to 45, wherein The control sample and the subject sample contain exosomes, and the exosomes contain PPP2R5C protein; Preferably, the exosomes are derived from neurons in plasma.

48. The use according to any one of claims 42 to 47, wherein The subject is a mammal, more preferably a human.

49. The use according to any one of claims 42 to 48, wherein The preparation is used for early diagnosis of Alzheimer's disease.

50. PPP2R5C protein for diagnosing, assisting in the diagnosis, detecting whether a subject has Alzheimer's disease, or for assessing the risk of a subject having Alzheimer's disease.

51. A method for diagnosing, assisting in the diagnosis, or detecting whether a subject has Alzheimer's disease, or assessing the risk of a subject having Alzheimer's disease, comprising: (i) detecting PPP2R5C protein in a sample from the subject using a reagent for detecting PPP2R5C protein; as well as (ii) comparing the PPP2R5C protein with a control sample, wherein the difference in the PPP2R5C protein compared to the control sample is used to diagnose, assist in diagnosis, detect whether a subject has Alzheimer's disease, or assess the risk of a subject having Alzheimer's disease.

52. The method of claim 51, wherein: The expression of the PPP2R5C protein in the subject's sample is reduced.

53. The method according to claim 51 or 52, wherein: The control sample and the subject sample are from plasma, brain tissue or cerebrospinal fluid.

54. The method of claim 51 or 52, wherein: The control sample and the subject sample contain exosomes, and the exosomes contain PPP2R5C protein; Preferably, the exosomes are derived from neurons in plasma.

55. The method according to any one of claims 51 to 54, wherein: The subject is a mammal, preferably a human.

56. The method according to any one of claims 51 to 55, wherein: The method is used for early diagnosis of Alzheimer's disease.

57. A kit, wherein: The kit is used for diagnosing, assisting in diagnosis, detecting whether a subject has Alzheimer's disease, or assessing the risk of a subject having Alzheimer's disease, and the kit contains a reagent for detecting the level of PPP2R5C.

58. The kit according to claim 57, wherein The PPP2R5C level is the protein level or mRNA level of PPP2R5C; Preferably, the kit further comprises a processing reagent for processing the biological sample; Preferably, the subject is a mammal, more preferably a human.

59. The kit according to claim 57 or 58, wherein The reagent for detecting the level of PPP2R5C comprises an antibody against the PPP2R5C protein; Preferably, the antibody is a monoclonal antibody or a polyclonal antibody; more preferably a monoclonal antibody.

60. The kit according to claim 57 or 58, wherein The reagent for detecting the level of PPP2R5C comprises a reagent for detecting the level of mRNA encoding PPP2R5C, preferably comprising primers and / or probes capable of quantifying the level of mRNA encoding PPP2R5C; Preferably, the reagent is used to measure the level of PPP2R5C by quantitative PCR.

61. The kit according to any one of claims 57 to 60, wherein The kit is used to distinguish between normal subjects and subjects suffering from Alzheimer's disease.

62. A drug screening method comprising: (i) administering an effective amount of the drug to be screened to a subject, and detecting the level of PPP2R5C protein in a sample from the subject using a reagent for detecting PPP2R5C protein; (ii) When the PPP2R5C protein level is increased compared with that before administration of the drug to be screened, the drug to be screened can be used to treat mild cognitive impairment and / or Alzheimer's disease.

63. A composition for detecting that a subject is in the preclinical stage of Alzheimer's disease, or for diagnosing, assisting in the diagnosis, detecting that a subject suffers from mild cognitive impairment, or for assessing the risk of a subject suffering from mild cognitive impairment, or for diagnosing, assisting in the diagnosis, detecting that a subject suffers from Alzheimer's disease, or for assessing the risk of a subject suffering from Alzheimer's disease, wherein: The composition comprises a reagent capable of detecting PPP2R5C protein.

64. A marker composition for detecting a subject in the preclinical stage of Alzheimer's disease, or for diagnosing, assisting in the diagnosis, detecting a subject suffering from mild cognitive impairment, or for assessing the risk of a subject suffering from mild cognitive impairment, or for diagnosing, assisting in the diagnosis, detecting a subject suffering from Alzheimer's disease, or for assessing the risk of a subject suffering from Alzheimer's disease, wherein: The marker composition contains PPP2R5C protein.

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