Alzheimer's disease early diagnosis marker and application thereof

Abstract

This invention discloses an early diagnostic biomarker for Alzheimer's disease (AD) and its application, belonging to the field of medical and pharmaceutical technology. Specifically, it discovers that the PPP2R5C protein in plasma neuronal-derived exosomes is an early diagnostic biomarker for AD. Based on this discovery, it also provides an application of the biomarker: an early AD diagnostic kit containing a monoclonal antibody against the PPP2R5C protein. This invention overcomes the detection limitations of lumbar puncture and PET-CT methods used in the early diagnosis of AD. Experimental results show that PPP2R5C in plasma neuronal-derived exosomes is significantly downregulated in both AD patients and patients with amnestic mild cognitive impairment (aMCI), exhibiting a trend of normal cognitive function controls > aMCI > AD. This demonstrates that PPP2R5C in plasma neuronal-derived exosomes is an early diagnostic biomarker for AD, and provides the application of an early AD diagnostic kit based on this PPP2R5C biomarker.

Description

Technical Field

[0001] This invention belongs to the field of medical and pharmaceutical technology, and in particular relates to an early diagnostic biomarker for Alzheimer's disease and its application. Background Technology

[0002] Despite recent progress in understanding the molecular mechanisms of Alzheimer's disease (AD), the prospects for AD treatment remain uncertain. The selection of therapeutic targets and the timing of intervention are crucial factors influencing the success of AD etiological treatment, with patients in the early stages of the disease benefiting more from such treatment. Currently, the main diagnostic methods for AD include lumbar puncture for cerebrospinal fluid analysis and PET-CT scans. However, lumbar puncture is an invasive procedure with inherent risks, resulting in low acceptance as a screening method; PET-CT scans are expensive, carry radiation exposure risks, and are often unavailable in primary care hospitals, limiting their application. These drawbacks make preclinical diagnosis of AD difficult.

[0003] Currently, the diagnosis of preclinical Alzheimer's disease (AD) relies on biomarker detection. According to the diagnostic criteria published by the International Working Group (IWG2) in 2014, the preclinical stage of AD requires a decrease in Aβ1–42 levels in cerebrospinal fluid and an increase in tau protein levels or increased PET-PET (pill-in-place fibrous amyloid) retention. However, lumbar puncture is an invasive procedure with certain risks, resulting in low acceptance as a screening method; PET-CT (positron emission tomography) is expensive, carries radiation exposure risks, and is not readily available in primary hospitals, limiting its application. These drawbacks make the diagnosis of preclinical AD difficult to implement. Therefore, developing simple and reliable early screening methods is crucial for the early diagnosis and treatment of AD.

[0004] Studies on plasma neuronal-derived exosomes show good consistency, and their related proteins have significant application value in the early diagnosis, diagnosis, and differential diagnosis of Alzheimer's disease (AD). However, AD is a complex disease, and its pathogenesis is not limited to a single target or pathway; biological changes also exhibit this characteristic. Furthermore, previous etiological treatments targeting Aβ and tau proteins have all failed, suggesting the need to identify new AD disease pathways and biological targets. Previous studies on AD biomarkers and exosomes have mostly utilized ELISA methods to detect a few specific proteins, which cannot comprehensively reflect the relationship between neuronal-derived exosomal proteins and AD. Summary of the Invention

[0005] This invention provides an early diagnostic biomarker for Alzheimer's disease (AD) and its application, aiming to address the detection deficiencies of current lumbar puncture and PET-CT methods used for early AD diagnosis.

[0006] Current research on AD biomarkers and exosomes mostly utilizes the ELISA method to detect a few specific proteins, which cannot comprehensively reflect the relationship between plasma neuron-derived exosome proteins and AD.

[0007] This invention utilizes mass spectrometry to comprehensively analyze the plasma neuronal-derived exosome proteome of patients with familial Alzheimer's disease (AD) and presymptomatic AD as screening subjects during the screening period. This analysis identifies PPP2R5C as a potential early diagnostic marker for AD. Subsequently, patients with sporadic mild AD and amnestic mild cognitive impairment (aMCI) are used as validation subjects to determine the diagnostic efficacy of the candidate markers. The results show that PPP2R5C in plasma neuronal-derived exosomes can serve as an early diagnostic biomarker for AD.

[0008] In summary, PPP2R5C is a biomarker for the early diagnosis of AD.

[0009] Furthermore, PPP2R5C in plasma neuronal-derived exosomes is a biomarker for early diagnosis of AD.

[0010] The application of PPP2R5C, an early diagnostic biomarker for Alzheimer's disease (AD), specifically involves an early diagnostic kit for AD, comprising a monoclonal antibody against the PPP2R5C protein.

[0011] The PPP2R5C protein monoclonal antibody can specifically recognize the PPP2R5C protein and measure the content of PPP2R5C (an early diagnostic marker for AD) in the plasma neuronal-derived exosomes of the tested individuals, thereby enabling the early diagnosis of AD.

[0012] Furthermore, the AD early diagnostic kit also includes a plasma neuron-derived exosome extraction reagent, which includes Thrombin PlasmaPrep for Exosome Precipitation, Pierce Streptavidin Agarose Resin, BSA, Exosomes Precipitation Solution, and Anti-Human CD171 Biotin.

[0013] The objective of this invention is achieved through the following technical solution:

[0014] This invention overcomes the detection defects of lumbar puncture and PET-CT detection methods used in the early diagnosis of Alzheimer's disease (AD). The experimental results of this invention show that the level of PPP2R5C protein in plasma neuronal-derived exosomes is significantly downregulated in AD patients, showing a trend of normal cognitive function control > aMCI > AD, thus proving that PPP2R5C in plasma neuronal-derived exosomes is a biomarker for the early diagnosis of AD, and providing the application of an AD early diagnostic kit based on this AD early diagnostic biomarker PPP2R5C. Attached Figure Description

[0015] Figure 1 This is a graph showing the subject characteristic curves of a normal control and aMCI patients constructed in Embodiment 2 of the present invention.

[0016] Figure 2 This is a graph showing the subject characteristic curves constructed in Example 2 of the present invention for normal controls and sporadic mild AD patients.

[0017] Figure 3 This is a plot of subject characteristic curves constructed in Embodiment 2 of the present invention for patients with sporadic mild AD and aMCI. Detailed Implementation

[0018] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0019] Furthermore, the technical solutions of the various embodiments can be combined with each other, but only if they are feasible for those skilled in the art. If the combination of technical solutions is contradictory or cannot be implemented, it should be considered that such combination of technical solutions does not exist and is not within the scope of protection claimed by this invention.

[0020] Currently, the main clinical diagnostic methods for Alzheimer's disease (AD) using lumbar puncture and PET-CT have the following drawbacks: lumbar puncture is an invasive procedure with certain risks, resulting in low acceptance as a screening method; PET-CT examinations are expensive, carry radiation exposure risks, and are not readily available in primary hospitals, limiting their application. To address these technical problems, this invention proposes an early diagnostic biomarker for AD and its application, aiming to solve the detection deficiencies of the current lumbar puncture and PET-CT methods used for early AD diagnosis.

[0021] Example 1

[0022] This study included 41 participants, including 13 patients with confirmed FAD (familial atopic dermatitis), 10 asymptomatic carriers of AD pathogenic gene mutations (pre-AD), and 18 healthy controls. To reduce intra-group variability, the samples were pooled. Based on different gene mutations, the 13 FAD patients were divided into 5 groups: two groups each carrying PSEN1 and PSEN2 gene mutations, one group carrying APP gene mutations, and 9 age- and sex-matched healthy controls were pooled into 3 groups. Similarly, the pre-AD group was divided into 5 groups: three groups carrying PSEN1 gene mutations, one group each carrying PSEN2 and APP gene mutations, and 9 age- and sex-matched healthy controls were pooled into 3 groups. A modified precipitation method was used to extract neuronal-derived exosomes from the plasma of the 41 participants. Label-free LC-MS / MS was used for qualitative, quantitative, and bioinformatics analysis of their proteomes to identify differentially expressed proteins common to both the FAD and pre-AD groups. The results showed that the expression level of PPP2R5C protein was higher in healthy individuals than in pre-AD individuals, and higher in AD individuals.

[0023] The improved precipitation method described above specifically includes the following steps:

[0024] 1.500ul of plasma, thawed at 4°C, with 5ul of protease inhibitor added;

[0025] 2. Add 5 μL Thrombin Plasma Prep for Exosome precipitation, mix well at room temperature for 5 min, 10000 g × 5 min;

[0026] 3. Take the supernatant, add 10ul Pierce Streptavidin Agarose Resin and 50ul 3% BSA, mix well, and incubate at 4°C for 5 hours;

[0027] 4. Add 126 μL of Exosomes Precipitation Solution to the supernatant, let stand for 1 hour, and then centrifuge for 30 minutes;

[0028] 5. After washing, add 250 μL PBS, 5 μL protease inhibitor, 50 μL 3% BSA, 4 μL Anti-Human CD171 Biotin and 25 μL Pierce Streptavidin Agarose Resin to the precipitate, and incubate overnight at 4°C.

[0029] 6. The next day, centrifuge and discard the supernatant; the precipitate is the neuronal-derived exosome.

[0030] The specific steps of the label-free LC-MS / MS technology used above are as follows:

[0031] Ten μg of exosomal protein samples obtained by the modified precipitation method were subjected to gradient Q-exactive analysis over a 1-hour period, followed by three LC-MS / MS analyses. The analysis included FASP enzymatic digestion, separation using Easy nLC at a nanoliter flow rate, and protein mass spectrometry analysis using a Q-Exactive mass spectrometer after HPLC separation. The mass spectrometry detection time was 2 hours, with positive ion detection. The scan range was set to 300–1800 m / z for the precursor ion. The resolution for the primary mass spectrometer was 70,000 at 200 m / z. The target value was set to 1e6, the maximum IT was 50 ms, and the dynamic exclusion time was 60.0 seconds. Database identification was performed using MaxQuant software (version 1.5.3.17).

[0032] Example 2

[0033] This study included 64 participants, including 20 patients with mild Alzheimer's disease (AD), 12 patients with aMCI (atypical mesenchymal disease), and 32 age- and sex-matched healthy controls. A modified precipitation method was used to extract neuronal-derived exosomal proteins from the plasma of all participants, and the PRM method was used for quantification of PPP2R5C. The expression levels of candidate proteins were compared among mild AD / healthy controls, aMCI / healthy controls, and mild AD / aMCI to screen for differentially expressed proteins. Receiver characteristic curves were constructed as shown below. Figures 1 to 3 As shown, comparing the diagnostic efficacy of different indicators for mild AD and aMCI, it was found that PPP2R5C in plasma neuronal-derived exosomes may serve as an indicator for early diagnosis of AD, and may change before symptoms appear.

[0034] The PRM method specifically involves the following steps: During the PRM method establishment phase, three samples (20 μg protein per sample) were randomly selected from each group and subjected to enzymatic digestion using LysC + Trypsin. The digested products were desalted, lyophilized, and the peptides were reconstituted with 0.1% FA. The peptide concentration was then determined using OD280 for mass spectrometry analysis. A suitable amount of the digested peptides from each group was taken, and the 12 samples were mixed into four samples: P_mix, C for P_mix, MCI_mix, and C for MCI_mix. Based on the sequences of 26 target proteins and previous label-free results, specific peptides for the target proteins were initially selected for subsequent PRM detection. Four mixed samples were analyzed by LC-MS / MS, with a mass spectrometry analysis time of 2 hours (2*2hr). Mass spectrometry data were retrieved from the database using Maxquant 1.5.3.17 and imported into Skyline software to initially screen for reliable peptides representing the target protein and to compile a list of proteins suitable for PRM quantification. The PRM method parameters were optimized to determine the LC-PRM / MS mass spectrometry method. Appropriate amounts of peptides were taken from the four mixed samples, and an appropriate amount of AQUA internal standard peptide was incorporated. LC-PRM / MS mass spectrometry was performed on each sample for 1 hour, repeated three times. Skyline was used to analyze the data and select peptides and daughter ions for protein quantification.

[0035] PPP2R5C in plasma neuronal-derived exosomes was significantly downregulated in sporadic mild AD patients, showing a trend of normal cognitive function controls > aMCI > AD, suggesting that PPP2R5C is an early screening biomarker for AD.

[0036] This embodiment utilizes mass spectrometry analysis to screen patients with familial Alzheimer's disease (AD) and presymptomatic AD. A comprehensive analysis of the plasma neuronal-derived exosome proteome was conducted to identify PPP2R5C as a potential early diagnostic marker for AD. Subsequently, patients with sporadic mild AD and amnestic mild cognitive impairment (aMCI) were used as validation subjects to determine the diagnostic efficacy of the candidate markers. The study found that PPP2R5C in plasma neuronal-derived exosomes can serve as an early diagnostic biomarker for AD.

[0037] In summary, PPP2R5C is a biomarker for the early diagnosis of AD.

[0038] Furthermore, PPP2R5C in plasma neuronal-derived exosomes is a biomarker for early diagnosis of AD.

[0039] Furthermore, the PPP2R5C protein in plasma neuronal-derived exosomes is a biomarker for early diagnosis of AD.

[0040] The application of PPP2R5C, an early diagnostic biomarker for Alzheimer's disease (AD), specifically involves an early diagnostic kit for AD, comprising a monoclonal antibody against the PPP2R5C protein.

[0041] The PPP2R5C protein monoclonal antibody can specifically recognize the PPP2R5C protein and measure the content of PPP2R5C (an early diagnostic marker for AD) in the plasma neuronal-derived exosomes of the tested individuals, thereby enabling the early diagnosis of AD.

[0042] Furthermore, the aforementioned early AD diagnostic kit also includes a plasma neuron-derived exosome extraction reagent; the plasma neuron-derived exosome extraction reagent includes Thrombin PlasmaPrep for Exosome Precipitation, Pierce Streptavidin Agarose Resin, BSA, Exosomes Precipitation Solution, and Anti-Human CD171 Biotin.

[0043] The objective of this invention is achieved through the following technical solution:

[0044] This invention overcomes the detection defects of lumbar puncture and PET-CT detection methods used in the early diagnosis of Alzheimer's disease (AD). The experimental results of this invention show that PPP2R5C in plasma neuronal-derived exosomes is significantly downregulated in AD patients, showing a trend of normal cognitive function control > aMCI > AD, thus proving that PPP2R5C in plasma neuronal-derived exosomes is a biomarker for the early diagnosis of AD, and providing the application of an AD early diagnostic kit based on this AD early diagnostic biomarker PPP2R5C.

[0045] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. Application of PPP2R5C protein in plasma neuron-derived exosomes as an early diagnostic marker for Alzheimer's disease in the preparation of a kit.

2. An early diagnostic kit for Alzheimer's disease, characterized by: The kit comprises a PPP2R5C protein monoclonal antibody and a plasma neuron-derived exosome extraction reagent.

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