Application of ATP6V1E1 as Alzheimer disease diagnosis target

Abstract

The invention relates to the technical field of disease target research, in particular to application of ATP6V1E1 as an Alzheimer's disease diagnosis target, and discloses application of ATP6V1E1 as a disease marker in preparation of a reagent and / or a medicine for diagnosing and / or treating Alzheimer's disease. The gene sequence of the ATP6V1E1 is as shown in SEQ ID NO. 1. The ATP6V1E1 gene can promote the autophagy function and improve the Alzheimer's disease caused by cognitive impairment due to Tau accumulation. Important clinical significance is provided for exploring a potential target spot for treating AD, and wide application prospects are achieved.

Description

Technical Field

[0001] The present invention relates to the technical field of disease target research, and in particular to the application of ATP6V1E1 as a diagnostic target for Alzheimer's disease. Background Art

[0002] Alzheimer's disease (AD) is a degenerative disease of the central nervous system that occurs in the elderly and pre-elderly stages. In the early stages of the disease, patients often show symptoms of cognitive impairment, such as memory loss and confusion. According to statistics, the number of dementia patients worldwide has exceeded tens of millions, and is showing a rapid growth trend. Among them, AD is the most common type of dementia. One of the two typical pathological characteristics of AD is the neurofibrillary tangles formed by the aggregation of Tau proteins. Autophagy, as an important self-protection mechanism of cells, plays an important role in clearing aggregated pathological proteins. The latest studies have shown that lysosomal acidification disorders during autophagy are closely related to the pathogenesis of AD. The hydrolases in lysosomes need to be in an acidic environment to perform their digestion and decomposition functions. Lysosomal acidification disorders will hinder the degradation of abnormal proteins, thereby exacerbating the aggregation of neurofibrillary tangles in the patient's brain. Therefore, exploring the key molecules that affect lysosomal acidification in the autophagy pathway is of great significance for screening early biomarkers and related therapeutic targets for diagnosing AD;

[0003] V-type proton ATPase (v-ATPase) can pump H+ ions from the cytoplasm into the lysosome. Therefore, the maintenance of the acidic environment in the lysosome mainly depends on v-ATPase. Once the lysosomal acidification is impaired, the waste decomposition pipeline will stagnate, causing a large number of waste intermediates to form and accumulate in the lysosome, including β-amyloid protein and its precursor protein; the pathogenesis of AD is mainly related to abnormal neurofibrillary tangles caused by the deposition of β-amyloid protein (Aβ) and the hyperphosphorylation of microtubule-associated protein Tau protein. In addition, AD is also closely related to mitochondrial dysfunction, energy metabolism disorders and neuroinflammation.

[0004] The existing effective diagnostic and treatment methods for AD are very limited. Currently, it can only be alleviated clinically by using cholinesterase inhibitors, N-methyl-D-aspartate receptor antagonists, drugs that improve brain metabolism and neuroprotective drugs.

[0005] Since the main pathological amyloid plaques of AD (mainly composed of extracellular deposited Aβ) and neurofibrillary tangles (mainly intracellular hyperphosphorylated Tau) may have occurred before the onset of clinical cognitive impairment, effective therapeutic drugs targeting Aβ and Tau have not achieved satisfactory results. Therefore, the research on AD urgently needs to explore new pathogenesis and therapeutic targets. Summary of the invention

[0006] The purpose of the present invention is to address the problems existing in the background technology and propose the application of ATP6V1E1 as a diagnostic target for Alzheimer's disease.

[0007] The technical solution of the present invention is the use of ATP6V1E1 as a disease marker in the preparation of reagents and / or drugs for diagnosing and / or treating Alzheimer's disease.

[0008] The gene sequence of ATP6V1E1 is shown in SEQ ID NO.1.

[0009] The ATP6V1E1 gene promotes autophagy function and improves Alzheimer's disease caused by cognitive damage due to Tau accumulation.

[0010] A kit for detecting Alzheimer's disease comprises a reagent for detecting the content of ATP6V1E1 in a biological sample.

[0011] Preferably, the kit detects the content of ATP6V1E1 protein in peripheral blood cells of a biological sample.

[0012] A drug combination comprising apigenin isoliquiritigenin C 26 H 30 O 13 , and pharmaceutically acceptable excipients.

[0013] Compared with the prior art, the present invention has the following beneficial technical effects:

[0014] The ATP6V1E1 screened out by the present invention can be used as a disease marker for diagnosing Alzheimer's disease; or a kit can be prepared by using ATP6V1E1 as a detection target to detect the ATP6V1E1 protein content in peripheral blood cells of biological samples. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 The efficiency diagram for distinguishing normal and cognitively impaired people based on the expression of ATP6V1E1 in monocytes;

[0016] Figure 2 is the expression level of ATP6V1E1 in AD brain tissue;

[0017] Figure 3 This is a graph showing a significant correlation between decreased ATP6V1E1 expression and Tau pathological grade and decreased v-ATPase enzyme activity level;

[0018] Figure 4 Overexpression of ATP6V1E1 improves autophagic flux and tau pathology in HEK293-tau cells.

[0019] Figure 5Ratio curve ranking of 153 drug compounds for molecular docking analysis. DETAILED DESCRIPTION

[0020] Example 1

[0021] In order to establish effective peripheral diagnostic biomarkers and explore potential therapeutic targets for AD:

[0022] This example systematically integrates brain tissue and peripheral monocyte omics data using bioinformatics technology, analyzes the molecular modules of central and peripheral linkage of cognitive impairment in the elderly, and confirms the value of potential biomarkers in early diagnosis of AD by expanding the population and longitudinal tracking. When analyzing brain tissue and peripheral monocyte transcriptomic data, it was found that the V-type proton ATPase subunit E1 (ATP6V1E1) was significantly downregulated in the central and peripheral of AD patients (see Basic Figure 1 and 2 ), and conduct further exploration on this basis.

[0023] Using multi-omics analysis techniques, Figure 1 As shown in A and D, the expression of ATP6V1E1 in monocytes is effective in distinguishing between normal and cognitively impaired people compared to the normal group. (A and D) Compared to the normal group, ATP6V1E1 is significantly reduced in monocytes of AD patients, and the asterisk represents a significant correlation: ****p<0.0001. In peripheral blood cells, it was found that the lysosomal ion channel-related protein ATP6V1E1 showed a consistent decrease in mild cognitive impairment (MCI) and AD patients (see Basic Figure 1 ); Logistic regression analysis was performed to draw the area under the curve to evaluate the efficiency of ATP6V1E1 in distinguishing normal and cognitively impaired people. Figure 1 In BC and EF, the area under the ROC curve (AUC) analysis in two different AD cohorts showed that changes in ATP6V1E1 in monocytes can effectively distinguish normal and cognitively impaired subjects: in the GSE63060 cohort, the AUC was 0.759, the sensitivity was 0.916, and the specificity was 0.481; in the GSE63061 cohort, the AUC was 0.677, the sensitivity was 0.739, and the specificity was 0.540 (see Basic Figure 1 The above results indicate that decreased expression of ATP6V1E1 may be closely related to cognitive impairment and can be used as a candidate biomarker for AD diagnosis.

[0024] To clarify the correlation between decreased ATP6V1E1 protein levels and learning and memory disorders caused by tau accumulation: the learning and memory abilities of P301L tau model mice aged 3, 6, 9, 12, and 18 months were tested; hippocampal tissue samples were then taken for protein blotting and immunofluorescence experiments of ATP6V1E1; the levels of phosphorylation of Tau protein, a classic AD pathology, were detected using relevant immunoblotting; finally, the levels of relevant tau proteins were correlated with the expression of ATP6V1E1 to clarify the relationship between ATP6V1E1 and Tau pathology.

[0025] To clarify the correlation between ATP6V1E1 and synaptic plasticity-related indicators: hippocampal tissue samples of P301L mice of the above-mentioned ages were taken for a. Golgi staining experiments to detect the length of neuronal axons and dendrites, total dendritic spine density, and the number of mushroom-shaped dendritic spines. b. Electrophysiological detection of I / O curves, PPR, and LTP; c. Electron microscopy to observe the morphology, size, and number of synaptic vesicles; d. Immunofluorescence and protein immunoblotting experiments were used to analyze and detect the levels of primary neuronal MAP2 and synaptic-related proteins (Syn1, PSD95, GluN2A, GluN2B); e. Nissl staining to assess the number of neurons.

[0026] Pearson analysis was performed to observe the correlation between ATP6V1E1 protein levels and synaptic plasticity and learning and memory-related indicators in the process of learning and memory impairment caused by tau accumulation.

[0027] To explore the molecular mechanism by which upregulating ATP6V1E1 promotes autophagy and improves cognitive impairment caused by tau accumulation: HEK293 cell lines transfected with Lenti-siATP6V1E1-CFP and Lenti-CFP lentiviruses, tau-treated HEK293 cells, tau-treated HEK293 cells transfected with rAAV-ATP6V1E1-CFP adeno-associated virus, and simultaneously transfected with mCherry-GFP-LC3, as well as hippocampal tissue samples from the above groups were collected, and the lysosomal pH and v-ATPase enzyme activity levels were detected, and the correlation analysis between the enzyme activity levels of ATP6V1E1 and v-ATPase was analyzed; the protein levels of lysosomal-related proteins CTSD and Lamp1 were detected at the same time; the activity of CTSD was quantified by detecting the hydrolysis of the CTSD-specific fluorescent substrate Mca-GKPILFFRLK-Dnp; the number of lysosomes was detected using transmission electron microscopy, p62 immunoelectron microscopy was performed, and the number of p62 in autophagic lysosomes was observed. Figure 3 shown.

[0028] To explore the molecular mechanism by which tau causes decreased expression of ATP6V1E1:

[0029] (1) Detection of ATP6V1E1 mRNA level: HEK293-P301L tau cells, primary neurons (from P301L fetal mice), and P301L whole body samples were taken and the changes in ATP6V1E1 mRNA level were detected by fluorescence quantitative PCR technology;

[0030] (2) HEK293-P301L tau cells, primary neurons (from P301L fetal mice), and P301L whole body samples were taken, nuclear proteins were extracted, and transcription factor high-throughput screening methods were used to detect transcription factor activity and identify significantly changed transcription factors;

[0031] (3) Bioinformatics tools were used to analyze the transcription factors with specific binding sites to the promoter region of ATP6V1E1, and staining immunoprecipitation (CHIP) was used to identify the transcription factors that bind to the promoter region of ATP6V1E1. Finally, a transcription factor luciferase reporter vector was used to verify the activity of the transcription factors screened above.

[0032] (4) To determine the upstream transcription factors of ATP6V1E1, HEK293-Htau cells, primary neurons (from P301L fetal mice), and P301L whole samples were taken, and the protein levels of the target transcription factors were detected by western blotting. Then, primers were designed based on the promoter region of ATP6V1E1 and PCR was used to synthesize the fragment. The PCR product was connected to the pGL-Basic vector (Promega) to construct the pGL-Luc reporter, which was transfected into HEK293-Htau cells and primary neurons (from P301L fetal mice). The expression level of the transcription factors was upregulated or downregulated, and the levels of luciferas, mRNA and protein of ATP6V1E1 were detected. Finally, the transcription factors that regulate the transcription of ATP6V1E1 were determined.

[0033] According to the above research, ATP6V1E1 can be used as a disease marker in the preparation of reagents and / or drugs for diagnosing and / or treating Alzheimer's disease. The gene sequence of ATP6V1E1 is shown in SEQ ID NO.1.

[0034] The ATP6V1E1 gene promotes autophagy function and improves Alzheimer's disease caused by cognitive impairment due to Tau accumulation.

[0035] A kit for detecting Alzheimer's disease comprises a reagent for detecting the content of ATP6V1E1 in a biological sample; the kit detects the content of ATP6V1E1 protein in peripheral blood cells of the biological sample.

[0036] This project combines AD population database analysis and molecular biological mechanism exploration methods, starting from the theory that autophagy-lysosome dysfunction is the pathogenesis of AD, to explore the key role of ATP6V1E1 in cognitive impairment caused by tau accumulation, which is expected to provide new insights for AD drug development.

[0037] This project will explore in depth the molecular mechanism by which tau accumulation downregulates the expression of ATP6V1E1, leading to autophagy-lysosome disorder and cognitive impairment; clarify the molecular mechanism by which tau causes the decrease in ATP6V1E1 expression. The research results will provide a new theory for revealing the pathogenesis of AD.

[0038] like Figure 2 As shown, the expression level of ATP6V1E1 in AD brain tissue. (AD) Compared with the normal group, the entorhinal cortex, hippocampus, frontal cortex, and temporal cortex of AD patients were significantly reduced. Asterisks represent significant correlations: *p<0.05, ***p<0.001, ****p<0.0001. (EH) ATP6V1E1 showed high diagnostic efficiency in the entorhinal cortex, hippocampus, frontal cortex, and temporal cortex of AD, with an AUC value of 0.616-0.817

[0039] like Figure 3 As shown, decreased ATP6V1E1 expression was significantly correlated with decreased Tau pathology grade and v-ATPase enzyme activity level. (AB) In human brain tissue, ATP6V1E1 expression was significantly negatively correlated with Tau pathology grade. Among them, Tau pathology grade includes neurofibrillary tangles (NTF) and Braak score. (C) Compared with wild-type mice, ATP6V1E1 was significantly reduced in the hippocampus of P301L mice. (D) Compared with wild-type mice, v-ATPase enzyme activity level was significantly reduced in the hippocampus of P301L mice. (E) In the hippocampus of P301L mice, ATP6V1E1 expression was significantly positively correlated with v-ATPase enzyme activity level. Asterisks represent significant correlations: ****p<0.0001.

[0040] like Figure 4 As shown, overexpression of ATP6V1E1 improves autophagic flux and tau pathology in HEK293-tau cells. (A) Overexpression of ATP6V1E1 improves autophagic flux in HEK293-tau (HEK293-P301L tau) cells. (B-D) Overexpression of ATP6V1E1 improves the level of phosphorylated tau S404 (p-Tau S404, pS404) in HEK293-tau cells. Asterisks represent significant correlations: ****p<0.0001.

[0041] The embodiments of the present invention are described in detail above with reference to the accompanying drawings, but the present invention is not limited thereto, and various changes can be made within the knowledge scope of technicians in the relevant technical field without departing from the purpose of the present invention.

[0042] Example 2

[0043] Design a kit for detecting Alzheimer's disease, which is used to detect the content of ATP6V1E1 in biological samples:

[0044] ATP6V1E1 is an important protein located in cells and is involved in a variety of physiological processes, such as cell signal transduction and material transport. Accurate detection of its content is of great significance for studying cell function and disease diagnosis. Enzyme-linked immunosorbent assay (ELISA) is a commonly used and sensitive detection method suitable for quantitative analysis of specific proteins in biological samples. This design scheme aims to develop an ELISA kit for detecting the content of ATP6V1E1.

[0045] The design of the ELISA kit is based on the principle of antigen-antibody reaction. First, the specific antibody is fixed on the microplate, and the sample to be tested is added to allow the ATP6V1E1 in the sample to bind to the antibody. Then, the enzyme-labeled antibody is added to react with the bound ATP6V1E1. Finally, the substrate is added, and the enzyme catalyzes the substrate to produce a color change, thereby quantitatively detecting the content of ATP6V1E1.

[0046] (1) Kit composition

[0047] Microplate: pre-coated with an antibody that specifically recognizes ATP6V1E1.

[0048] Standard: ATP6V1E1 with known concentration, used to draw the standard curve.

[0049] Enzyme-labeled antibody: an antibody that specifically binds to ATP6V1E1 and is labeled with horseradish peroxidase (HRP).

[0050] Substrate solution: such as TMB (3,3',5,5'-tetramethylbenzidine), produces a blue product under HRP catalysis.

[0051] Stop solution: such as sulfuric acid solution, used to terminate the enzymatic reaction and convert the blue product into yellow to facilitate absorbance detection.

[0052] Washing solution: used to clean the microplate and remove non-specifically bound substances.

[0053] Sample diluent: used to dilute the sample to be tested to ensure that the concentration of ATP6V1E1 in the sample is within the detection range.

[0054] (2) Operation steps

[0055] Sample preparation: Dilute the sample to be tested in a certain ratio to ensure that the concentration of ATP6V1E1 in the sample is within the detection range.

[0056] Sample addition: Add the standard and diluted samples to the microplate pre-coated with antibodies, set up duplicate wells for each sample, and incubate at 37°C for 1 hour.

[0057] Washing: Pour out the liquid in the wells and wash the microplate 3 times with washing solution, leaving it for 1 minute each time.

[0058] Add enzyme-labeled antibody: Add enzyme-labeled antibody and incubate at 37°C for 30 minutes.

[0059] Wash: Repeat step 3.

[0060] Add substrate solution: Add substrate solution and incubate at 37°C in dark for 15 minutes.

[0061] Stop reaction: Add stop solution to terminate the enzymatic reaction.

[0062] Reading: Use an ELISA reader to measure the absorbance value (OD value) of each well at a wavelength of 450 nm.

[0063] (3) Data processing

[0064] Draw a standard curve: Draw a standard curve with the concentration of the standard as the horizontal axis and the corresponding OD value as the vertical axis.

[0065] Calculate sample concentration: According to the OD value of the sample, find the corresponding ATP6V1E1 concentration on the standard curve.

[0066] Sample processing: Ensure that the samples are fresh and avoid repeated freezing and thawing to avoid affecting the test results.

[0067] Operating specifications: Strictly follow the instructions to avoid cross contamination and operating errors.

[0068] Temperature control: Ensure accurate incubation temperature and time to ensure the sensitivity and specificity of the reaction.

[0069] Protect substrate from light: The substrate solution needs to be stored in a dark place and avoid direct sunlight when using.

[0070] Example 3

[0071] A library of 153 drug compounds was screened for a drug that could increase the protein content of ATP6V1E1.

[0072] 1. Literature research and concentration optimization: First, a detailed literature search was conducted for each drug compound to determine its optimal concentration when acting on HEK293T cells. This step ensures the accuracy and effectiveness of subsequent experiments.

[0073] 2. Uniform drug administration to cells stably expressing MAPT: Based on the results of literature research, the optimized drug concentration was uniformly applied to cell lines stably expressing MAPT (microtubule-associated protein tau) to simulate disease-related environments and provide a reliable basis for subsequent analysis.

[0074] 3. Collection of cell culture supernatant: 24 hours after drug treatment, carefully collect the cell culture supernatant. This step is crucial for subsequent biomarker detection.

[0075] 4. ELISA kit verification and Ratio curve drawing: Use ELISA (enzyme-linked immunosorbent assay) kit to quantitatively analyze specific molecules or biomarkers in the supernatant. According to the test results, draw a Ratio curve, which can intuitively reflect the changes in the expression level of the target molecule under different drug treatments.

[0076] 5. Molecular docking analysis: To further understand the interaction mechanism between drugs and potential targets, molecular docking simulation is performed. This step predicts the binding ability of drug molecules with biological macromolecules (such as proteins) through computational means, providing a theoretical basis for drug screening.

[0077] 6. Comprehensive evaluation and optimal selection: Finally, a comprehensive evaluation was conducted by combining the ranking information of the Ratio curve and the activation energy data obtained by molecular docking analysis. After rigorous screening, Isoliquiritinapioside was identified as the most promising candidate drug, and its molecular formula is as follows:

[0078]

[0079] It exhibits optimal performance in regulating the expression of target molecules and binding to targets.

[0080] Through the above meticulous and systematic steps, not only the scientificity and rigor of the screening process are ensured, but also a solid foundation is laid for subsequent drug research and development work.

[0081] The embodiments of the present invention are described in detail above with reference to the accompanying drawings, but the present invention is not limited thereto, and various changes can be made within the knowledge scope of technicians in the relevant technical field without departing from the purpose of the present invention.

Claims

1. Use of ATP6V1E1 as a disease marker in the preparation of reagents and / or drugs for diagnosing and / or treating Alzheimer's disease.

2. The use according to claim 1, characterized in that The gene sequence of ATP6V1E1 is shown in SEQ ID NO.

1.

3. The use according to claim 1, characterized in that The ATP6V1E1 gene promotes autophagy function and improves Alzheimer's disease caused by cognitive damage due to Tau accumulation.

4. A kit for detecting Alzheimer's disease, characterized in that: Included are reagents for detecting the amount of ATP6V1E1 in a biological sample.

5. The kit for detecting Alzheimer's disease according to claim 4, characterized in that: The kit detects the content of ATP6V1E1 protein in peripheral blood cells of a biological sample.

6. A drug combination, characterized in that Including apigenin isoliquiritigenin C 26 H 30 O 13 , and pharmaceutically acceptable excipients.

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