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WO2024240079A1 - Marqueur de détection, marqueur de diagnostic, leur utilisation et kit - Google Patents

Marqueur de détection, marqueur de diagnostic, leur utilisation et kit Download PDF

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Publication number
WO2024240079A1
WO2024240079A1 PCT/CN2024/093898 CN2024093898W WO2024240079A1 WO 2024240079 A1 WO2024240079 A1 WO 2024240079A1 CN 2024093898 W CN2024093898 W CN 2024093898W WO 2024240079 A1 WO2024240079 A1 WO 2024240079A1
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subject
ppp2r5c
alzheimer
disease
protein
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English (en)
Chinese (zh)
Inventor
沈璐
刘慧�
焦彬
张振涛
罗世林
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Xiangya Hospital of Central South University
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Xiangya Hospital of Central South University
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Priority to CN202480004008.7A priority Critical patent/CN119895264A/zh
Publication of WO2024240079A1 publication Critical patent/WO2024240079A1/fr
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • G01N33/6896—Neurological disorders, e.g. Alzheimer's disease
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
    • G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/28—Neurological disorders
    • G01N2800/2814—Dementia; Cognitive disorders
    • G01N2800/2821—Alzheimer

Definitions

  • 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 uses thereof, and corresponding kits; it also relates to a method for drug screening using PPP2R5C protein, a composition comprising a reagent capable of detecting PPP2R5C protein, and a marker composition containing PPP2R5C protein.
  • AD Alzheimer’s disease
  • pre-AD preclinical stage
  • MCI mild cognitive impairment
  • This stage can last for more than 20 years. It takes an average of 2-6 years from MCI to mild AD, and 8-10 years from mild to severe AD.
  • the AT(N) framework has developed AD from a clinical biological diagnosis to a pure biological definition separated from the clinical phenotype, which has caused controversy and challenges in daily clinical applications.
  • the diagnosis of AD still needs to be combined with biomarkers on the basis of clinical phenotypes.
  • cerebrospinal fluid (CSF) testing is highly invasive, and patients need to endure the pain of lumbar puncture.
  • Positron Emission Tomography-Computed Tomograph (PET-CT) examinations are limited by the level of regional economic development. Few hospitals can carry out the examinations, and the examinations are expensive and involve radiation. These factors limit the widespread application of cerebrospinal fluid testing and PET-CT examinations in clinical practice, and they are not suitable for early screening and dynamic evaluation.
  • AD Alzheimer's disease
  • plasma proteins may also undergo AD-specific changes. Therefore, clarifying changes in plasma proteins may provide more clues to understanding the pathogenesis of AD, and may also help with early screening and diagnosis of AD.
  • PET-CT examinations and cerebrospinal fluid tests blood tests have always attracted attention due to their advantages such as minimally invasive, convenient, low cost, and repeatability. When used in combination with other clinical information, they are expected to be used for early identification and dynamic evaluation of AD patients, as well as risk assessment of normal people.
  • the present invention provides detection markers for the preclinical stage of Alzheimer's disease, diagnostic markers for mild cognitive impairment, AD diagnostic markers and uses thereof, and corresponding kits, aiming to solve the detection defects existing in the current early diagnosis of AD and fill the gap in detection methods before the AD stage.
  • the inventors of the present invention used the method of mass spectrometry omics analysis, with AT (N) biomarker positive or evidence of pathogenic gene mutations and clear clinical diagnosis of AD patients (AD group), familial AD preclinical stage patients carrying AD pathogenic gene mutations and clear genetic diagnosis (pre-AD group) and healthy control group (CN) as screening research subjects, and conducted a comprehensive analysis of neuron-derived exosome proteins in the three groups of plasma, screened out the early diagnostic indicator PPP2R5C, and then used sporadic AD and mild cognitive impairment (MCI) patients as validation research subjects to determine the diagnostic efficacy of candidate indicators. It was found that the level of PPP2R5C was gradually downregulated in MCI and AD patients, showing a trend of healthy control>MCI>AD.
  • 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.
  • the inventors found through research that PPP2R5C reduces both total Tau and phosphorylated Tau in neurons, resulting in a reduction in toxic proteins of neurofibrillary tangles (NFTs) in neurons, thereby protecting the activity of neurons and reducing the risk of AD occurrence and development.
  • NFTs neurofibrillary tangles
  • a decrease in PPP2R5C will increase total Tau and phosphorylated Tau, increase the toxic proteins of neurofibrillary tangles formed in neurons, lead to neuronal death, and induce the occurrence of AD.
  • the PPP2R5C described in the present invention belongs to the phosphatase 2A (PP2A) regulatory subunit B family. It is also known as B56G, B56 ⁇ and PR6G.
  • PP2A is a major serine/threonine phosphatase that plays an important role in the balance of phosphorylation signals, affects the phosphorylation state of many proteins, and is related to the regulation of some signal transduction pathways, cell proliferation and differentiation, cell malignant transformation, cell cycle regulation, DNA replication/transcription and embryonic development.
  • PP2A consists of three parts, namely, the catalytic subunit (catalytic subunit C), the structural subunit (structural subunit) and the regulatory subunit (regulatory subunit B).
  • the A and C subunits combine to form a heterodimer in the cell, and AC is the core enzyme of PP2A. They can bind to the B subunit to form a heterotrimeric PP2A holoenzyme.
  • Regulatory subunit B contains 4 different family members, including B (B55/PR56), B' (B56/PR56), B" (PR48) and B"' (PR93/110).
  • the B' subunit has the greatest diversity, including ⁇ (PPP2R5A), ⁇ (PPP2R5B), ⁇ (PPP2R5C), ⁇ (PPP2R5D) and ⁇ (PPP2R5E).
  • the PPP2R5C of the present invention can be one of the multiple isomers of the PPP2R5C protein, or a combination of two or more of these isomers.
  • the amino acid sequence of the PPP2R5C is shown in SEQ ID NO: 3.
  • the PPP2R5C having this amino acid sequence is only an example of one of the isomers.
  • the present invention provides a detection marker for the preclinical stage of Alzheimer's disease, wherein the detection marker is PPP2R5C protein.
  • the subject in the preclinical stage of Alzheimer's disease carries an Alzheimer's disease-causing gene mutation
  • 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.
  • 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.
  • the subject in the preclinical stage of Alzheimer's disease carries a mutation in a gene that causes Alzheimer's disease.
  • the PPP2R5C protein is differentially expressed in a subject sample compared to a control sample.
  • 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.
  • the expression of the PPP2R5C protein in the subject sample in the preclinical stage of Alzheimer's disease is lower than that in the control sample from a normal subject, and higher than that in the subject sample with mild cognitive impairment.
  • control sample and the subject sample are from plasma, brain tissue, or cerebrospinal fluid.
  • control sample and the subject sample comprise exosomes, wherein the exosomes comprise PPP2R5C protein.
  • the exosomes are derived from neurons in plasma.
  • the subject is a mammal, preferably a human.
  • the present invention provides a PPP2R5C protein for detecting whether a subject is in the preclinical stage of Alzheimer's disease.
  • the present invention provides a method for detecting whether a subject is in the preclinical stage of Alzheimer's disease, comprising:
  • the expression of the PPP2R5C protein in the subject's sample is reduced, and the subject is considered to be in the preclinical stage of Alzheimer's disease.
  • control sample and the subject sample are from plasma, brain tissue, or cerebrospinal fluid.
  • control sample and the subject sample comprise exosomes, wherein the exosomes comprise PPP2R5C protein.
  • the exosomes are derived from neurons in plasma.
  • the subject is a mammal, preferably a human.
  • 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.
  • the PPP2R5C level is the protein level or mRNA level of PPP2R5C.
  • the reagent detects the level of PPP2R5C by the following methods: chromatography and/or mass spectrometry, fluorescence measurement, electrophoresis, immunoaffinity, hybridization, immunochemistry, ultraviolet spectroscopy, fluorescent powder, radiochemical analysis, near infrared spectroscopy, nuclear magnetic resonance spectroscopy, light scattering analysis and turbidimetry.
  • the reagent for detecting the level of PPP2R5C comprises an antibody against the PPP2R5C protein.
  • the antibody is a monoclonal antibody or a polyclonal antibody; more preferably, a monoclonal antibody.
  • 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.
  • the sequence of the primer used is shown in SEQ ID NO: 1-2.
  • the reagent measures PPP2R5C levels by quantitative PCR.
  • the kit further comprises a processing reagent for processing the biological sample.
  • the subject is a mammal, more preferably a human.
  • the kit is used to distinguish between a normal subject and a subject in a preclinical stage of Alzheimer's disease.
  • the present invention provides a diagnostic marker for mild cognitive impairment, wherein the diagnostic marker is PPP2R5C protein.
  • the mild cognitive impairment includes AD-induced mild cognitive impairment and non-AD-induced mild cognitive impairment.
  • the present invention provides the 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.
  • the PPP2R5C protein is differentially expressed in a subject sample compared to a control sample.
  • expression of the PPP2R5C protein is reduced in the subject's sample.
  • the expression of the PPP2R5C protein in patients with mild cognitive impairment is lower than that in subjects in the preclinical stage of Alzheimer's disease.
  • the expression of the PPP2R5C protein is higher in patients with mild cognitive impairment than in subjects with Alzheimer's disease.
  • control sample and the subject sample are from plasma, brain tissue, or cerebrospinal fluid.
  • control sample and the subject sample comprise exosomes, wherein the exosomes comprise PPP2R5C protein.
  • the exosomes are derived from neurons in plasma.
  • the subject is a mammal, preferably a human.
  • the present invention provides a method for diagnosing, assisting in the diagnosis, or detecting that a subject suffers from mild cognitive impairment, or assessing the risk of mild cognitive impairment in a subject, comprising:
  • expression of the PPP2R5C protein is reduced in the subject's sample.
  • control sample and the subject sample are from plasma, brain tissue, or cerebrospinal fluid.
  • control sample and the subject sample comprise exosomes, wherein the exosomes comprise PPP2R5C protein.
  • the exosomes are derived from neurons in plasma.
  • the subject is a mammal, preferably a human.
  • the present invention provides a kit for diagnosing, assisting in the diagnosis, 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.
  • the PPP2R5C level is the protein level or mRNA level of PPP2R5C.
  • the reagent detects the level of PPP2R5C by the following methods: chromatography and/or mass spectrometry, fluorescence measurement, electrophoresis, immunoaffinity, hybridization, immunochemistry, ultraviolet spectroscopy, fluorescent powder, radiochemical analysis, near infrared spectroscopy, nuclear magnetic resonance spectroscopy, light scattering analysis and turbidimetry.
  • the reagent for detecting the level of PPP2R5C comprises an antibody against the PPP2R5C protein.
  • the antibody is a monoclonal antibody or a polyclonal antibody; more preferably, a monoclonal antibody.
  • 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.
  • the sequence of the primer used is shown in SEQ ID NO: 1-2.
  • the reagent measures PPP2R5C levels by quantitative PCR.
  • the kit further comprises a processing reagent for processing the biological sample.
  • the subject is a mammal, more preferably a human.
  • the kit is used to distinguish between normal subjects and subjects with mild cognitive impairment.
  • the present invention provides a diagnostic marker for Alzheimer's disease, wherein the diagnostic marker is PPP2R5C protein.
  • the Alzheimer's disease includes familial AD and sporadic AD.
  • the Alzheimer's disease includes mild AD, moderate AD, and severe AD.
  • 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.
  • the PPP2R5C protein is differentially expressed in a subject sample compared to a control sample.
  • expression of the PPP2R5C protein is reduced in the subject's sample.
  • the expression of the PPP2R5C protein in patients with Alzheimer's disease is lower than that in patients with mild cognitive impairment.
  • the expression of the PPP2R5C protein is higher in patients with mild Alzheimer's disease than in patients with moderate Alzheimer's disease.
  • the expression of the PPP2R5C protein is higher in patients with moderate Alzheimer's disease than in patients with severe Alzheimer's disease.
  • control sample and the subject sample are from plasma, brain tissue, or cerebrospinal fluid.
  • control sample and the subject sample comprise exosomes, wherein the exosomes comprise PPP2R5C protein.
  • the exosomes are derived from neurons in plasma.
  • the subject is a mammal, preferably a human.
  • the formulation is used for early diagnosis of Alzheimer's disease.
  • the present invention provides a 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.
  • the present invention provides a method for diagnosing, assisting in the diagnosis, or detecting whether a subject suffers from Alzheimer's disease, or assessing the risk of a subject suffering from Alzheimer's disease, comprising:
  • expression of the PPP2R5C protein is reduced in the subject's sample.
  • control sample and the subject sample are from plasma, brain tissue, or cerebrospinal fluid.
  • control sample and the subject sample comprise exosomes, wherein the exosomes comprise PPP2R5C protein.
  • the exosomes are derived from neurons in plasma.
  • the subject is a mammal, preferably a human.
  • the method is used for early diagnosis of Alzheimer's disease.
  • the present invention provides a kit for diagnosing, assisting in the diagnosis, 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.
  • the PPP2R5C level is the protein level or mRNA level of PPP2R5C.
  • the reagent detects the level of PPP2R5C by the following methods: chromatography and/or mass spectrometry, fluorescence measurement, electrophoresis, immunoaffinity, hybridization, immunochemistry, ultraviolet spectroscopy, fluorescent powder, radiochemical analysis, near infrared spectroscopy, nuclear magnetic resonance spectroscopy, light scattering analysis and turbidimetry.
  • the reagent for detecting the level of PPP2R5C comprises an antibody against the PPP2R5C protein.
  • the antibody is a monoclonal antibody or a polyclonal antibody; more preferably, a monoclonal antibody.
  • 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.
  • the sequence of the primer used is shown in SEQ ID NO: 1-2.
  • the reagent measures PPP2R5C levels by quantitative PCR.
  • the kit further comprises a processing reagent for processing the biological sample.
  • the subject is a mammal, more preferably a human.
  • the kit is used to distinguish between a normal subject and a subject suffering from Alzheimer's disease.
  • the kit is used for early diagnosis of Alzheimer's disease.
  • the present invention provides a drug screening method comprising:
  • the drug to be screened can be used to treat mild cognitive impairment and/or Alzheimer's disease.
  • the present invention provides 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 has mild cognitive impairment, or for assessing the risk of a subject having mild cognitive impairment, or for diagnosing, assisting in the diagnosis, detecting that 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.
  • the subject in the preclinical stage of Alzheimer's disease carries a mutation in a gene causing Alzheimer's disease.
  • 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.
  • the subject in the preclinical stage of Alzheimer's disease carries a mutation in a gene causing Alzheimer's disease.
  • the present invention overcomes the detection defects of lumbar puncture and PET-CT detection methods used in early diagnosis of AD and the problem of 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.
  • 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.
  • FIG1 is a characterization diagram of neuron-derived exosomes in plasma in Example 1 of the present invention.
  • FIG2 is a diagram showing the mass spectrometry screening results of PPP2R5C in Example 2 of the present invention.
  • FIG3 is a diagram showing the efficacy validation results of the neuron-derived exosome PPP2R5C protein in plasma as an early diagnostic marker for AD in Example 3 of the present invention
  • FIG4 is a diagram showing the determination of PPP2R5C protein content 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;
  • FIG5 is a graph showing the WB detection results of PPP2R5C protein in human brain tissue in Example 6 of the present invention.
  • FIG6 is a diagram showing the results of the study of the intrinsic mechanism of PPP2R5C as an AD marker in Example 7 of the present invention, wherein “OE-PPP2R5C” indicates overexpression of PPP2R5C.
  • This example uses a modified precipitation method to extract neuronal exosomes from plasma, and the specific steps are as follows:
  • Plasma exosome extraction kit System Biosciences, Cat#EXOQ5A
  • 5 ⁇ L plasma exosome precipitation reagent Thrombin Plasma Prep for Exosome precipitation
  • the neuronal exosome-specific antibody CD171 (Abcam, #270455) and the exosome-specific antibody Alix (Abcam, #275377) were used to characterize neuronal exosomes by immunoblotting.
  • FIG. 1a The electron microscopic characterization of the morphology of neuron-derived exosomes in plasma in this example is shown in Figure 1a, the exosome particle size distribution diagram is shown in Figure 1b, and the specific characterization of exosomes belonging to neuron-derived exosomes is shown in Figure 1c.
  • the neuronal exosomes isolated from plasma have a typical morphology and are not ruptured.
  • the particle size distribution of neuronal exosomes isolated from plasma is uniform, with an average particle size of 107.3 nm.
  • the exosomes isolated from plasma are secreted by neurons.
  • pre-AD asymptomatic AD pathogenic gene mutation carriers
  • CN normal controls
  • the above samples were mixed within the group.
  • the 13 patients in the FAD group were mixed into 5 groups, including two groups carrying PSEN1 and PSEN2 gene mutations, one group carrying APP gene mutations, and 9 normal controls matched with age and gender were mixed into three groups.
  • Pre-AD was similarly divided into 5 groups, including three groups carrying PSEN1 gene mutations, one group carrying PSEN2 and APP gene mutations, and 9 normal controls matched with age and gender were mixed into three groups.
  • Plasma neuron-derived exosomes were extracted from 41 subjects, and their proteomes were qualitatively, quantitatively and bioinformatics clustering heat map analysis was performed using Label free LC-MS/MS technology to find the differentially expressed proteins shared by the FAD group and the pre-AD group, and the expression level of PPP2R5C protein was found to be CN>pre-AD>AD.
  • the resolution of the primary mass spectrometer was required to reach 70,000 at 200 m/z, 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 number 1.5.3.17) was used for library search and identification.
  • PPP2R5C protein number H0YN58
  • cluster group C represents CN
  • PER represents pre-AD
  • P represents the patient (patient) is the AD group.
  • 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 PPP2R5C was quantitatively detected by the parallel reaction monitoring (PRM) method.
  • PRM parallel reaction monitoring
  • the expression levels of candidate proteins were compared between mild AD/normal controls, MCI/normal controls, 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, and the diagnostic efficacy of different indicators for mild AD and MCI was compared. 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.
  • the selection criteria of the validation index are: According to the selection criteria of candidate early diagnostic biomarkers: 1. Differentially expressed proteins shared by FAD and pre-AD, and/or proteins related to dementia; 2. Meet the technical requirements of PRM detection, that is, each protein must have 2 or more unique peptides identified. The differentially expressed proteins and dementia-related proteins in Example 2 were screened, and finally a total of 26 proteins met the selection criteria and were further verified in the sporadic population.
  • the specific content of the PRM method is as follows: In the establishment stage of the PRM method, 4 samples were randomly selected from each group, 20 ⁇ g of protein in each sample, and proteolysis was performed according to LysC+Trypsin. The product after enzymatic hydrolysis was desalted and lyophilized, and the peptides were re-dissolved with 0.1% FA, and the peptide concentration was determined by OD280 for mass spectrometry testing. An appropriate amount of enzymatic hydrolysis peptides were taken from each group of samples, and 12 samples were mixed into 3 mixed samples within the group.
  • the specific peptides of the target protein were preliminarily selected for subsequent PRM detection.
  • LC-MS/MS analysis was performed on the three mixed samples, and the mass spectrometry analysis time was 2h (2*2hr); the mass spectrometry data was retrieved from the database using Maxquant1.5.3.17, and imported into Skyline software to preliminarily screen out credible peptides that can represent the target protein, and screen out a list of proteins that can be quantitatively analyzed by PRM.
  • the parameters of the PRM method were optimized to determine the LC-PRM/MS mass spectrometry method.
  • the ROC curve of PPP2R5C for distinguishing normal controls and MCI patients is shown in Figure 3a
  • the ROC curve of PPP2R5C for distinguishing normal controls and AD patients is shown in Figure 3b
  • the ROC curve of PPP2R5C for distinguishing MCI patients and AD patients is shown in Figure 3c.
  • PPP2R5C in neuronal exosomes in plasma has good diagnostic efficacy for distinguishing normal controls, MCI and AD, suggesting that PPP2R5C is an early screening marker for AD.
  • AD AD patients
  • MCI mild cognitive impairment
  • CN age- and sex-matched normal controls
  • Enzyme-linked immunosorbent assay was used to directly detect the content of PPP2R5C in plasma.
  • the ELISA kit used was produced by Abbexa, USA, with the catalog number abx500105.
  • the ELISA assay results in this example are shown in FIG4a
  • the efficacy verification analysis results are shown in FIG4b .
  • PPP2R5C in plasma can be used as an early screening marker for AD.
  • AD AD
  • MCI mild cognitive impairment
  • CN age- and gender-matched normal controls
  • qRT-PCR Real-time fluorescence quantitative reverse transcription polymerase chain reaction
  • h-PPP2R5C-F 5′-CAGCGAAGATTCTGCCCATCATG-3′(SEQ ID NO:1)
  • the qRT-PCR assay results in this example showed that compared with the normal control group (CN), the PPP2R5C mRNA level in plasma was 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 was consistent with the changes in the PPP2R5C protein level in the neuronal exosomes isolated from plasma and the direct measurement of the PPP2R5C protein level in plasma.
  • the WB measurement results in this example are shown in Figure 5a, and the results show that PPP2R5C is significantly reduced in the brain tissue of AD patients; as shown in the statistical results Figure 5b, both the elderly normal controls and the young normal controls have statistical differences with AD patients.
  • the above examples use mass spectrometry to analyze familial AD patients and pre-AD patients with clear genetic diagnosis as the research subjects in the screening period, and conduct a comprehensive analysis of their plasma neuron-derived exosome proteome to screen out the possible AD early diagnosis indicator PPP2R5C, and then use AD and mild cognitive impairment (MCI) patients as the research subjects in the verification period to determine the diagnostic efficacy of the candidate indicators, and find that PPP2R5C in plasma neuron-derived exosomes can be used as an AD early diagnosis marker.
  • MCI mild cognitive impairment
  • AD mild cognitive impairment
  • MCI mild cognitive impairment
  • ELISA and qRT-PCR detection methods were used to find that PPP2R5C protein in direct plasma can also be used as an AD early diagnosis marker; and human brain tissue WB detection shows that PPP2R5C is also significantly reduced in AD patients.
  • this example uses AD transgenic mice Tau P301S to isolate and culture primary hippocampal neurons of fetal mice.
  • adeno-associated viruses AAV-shPPP2R5C, AAV-PPP2R5C (Wuhan Shumi Brain Science Co., Ltd.) and corresponding control viruses are added to knock down or high-expression PPP2R5C expression in neurons.
  • WB immunoblotting
  • 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.

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  • Molecular Biology (AREA)
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Abstract

La présente invention relève du domaine technique des médicaments. L'invention concerne un marqueur de détection, un marqueur de diagnostic, leur utilisation, et un kit. Dans la présente invention, PPP2R5C est utilisé en tant que marqueur de détection du stade préclinique de la maladie d'Alzheimer (AD) et en tant que marqueur de diagnostic de la déficience cognitive légère et de l'AD ; ainsi, les problèmes de défauts de détection et de manque de biomarqueurs sanguins efficaces dans les méthodes de ponction lombaire et de détection PET-CT utilisées pour le diagnostic précoce de la maladie d'Alzheimer sont résolus.
PCT/CN2024/093898 2023-05-24 2024-05-17 Marqueur de détection, marqueur de diagnostic, leur utilisation et kit Pending WO2024240079A1 (fr)

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CN202310592108.3A CN116735892A (zh) 2023-05-24 2023-05-24 一种阿尔茨海默病早期诊断标志物及其应用

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CN116735892A (zh) * 2023-05-24 2023-09-12 中南大学湘雅医院 一种阿尔茨海默病早期诊断标志物及其应用
CN121114408A (zh) * 2024-08-05 2025-12-12 中南大学湘雅医院 一种基于呼出气中乙醇和吡咯作为标志物在制备诊断或者辅助诊断阿尔茨海默病产品中的应用

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