Application of ATP6V1D as Alzheimer disease diagnosis target

Abstract

The invention relates to the technical field of disease target research, in particular to application of ATP6V1D as an Alzheimer disease diagnosis target. The invention discloses application of ATP6V1D 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 ATP6V1D is as shown in SEQ ID NO. 1. The ATP6V1D 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 ATP6V1D 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 play a digestive and decomposing role. 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 lysosome 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. ATP6V1D It is an important subunit of the v-ATPase proton pump. Its mutation will lead to the assembly or stability of the v-ATPase enzyme complex, while its reduction will lead to an increase in lysosomal pH, resulting in a decrease in lysosomal degradation capacity. Although many AD-related diagnosis and treatment studies focus on the autophagy pathway, the specific mechanism of lysosomal acidification disorders is still unclear, and there has never been a report on the specific role of ATP6V1D in the progression of AD. Therefore, exploring the specific role of ATP6V1D in cognitive impairment caused by autophagy defects caused by lysosomal acidification disorders is of great significance for screening diagnostic and therapeutic targets for AD.

[0004] Based on the above problems, the present invention proposes an application of ATP6V1D as a diagnostic or therapeutic target for AD. Summary of the invention

[0005] The purpose of the present invention is to address the problems existing in the background technology and to propose an application of ATP6V1D as a diagnostic target for Alzheimer's disease.

[0006] The technical solution of the present invention is the use of ATP6V1D as a disease marker in the preparation of reagents and / or drugs for diagnosing and / or treating Alzheimer's disease.

[0007] The gene sequence of ATP6V1D is shown in SEQ ID NO.1.

[0008] The ATP6V1D gene promotes autophagy function and improves Alzheimer's disease caused by cognitive damage due to Tau accumulation.

[0009] A kit for detecting Alzheimer's disease, comprising a reagent for detecting the content of ATP6V1D in a biological sample.

[0010] The kit detects the content of ATP6V1D protein in peripheral blood cells of a biological sample.

[0011] A drug combination comprising apigenin isoliquiritigenin C 26 H 30 O 13 , and pharmaceutically acceptable excipients.

[0012] Compared with the prior art, the present invention has the following beneficial technical effects:

[0013] The ATP6V1D 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 ATP6V1D as a detection target to detect the ATP6V1D protein content in peripheral blood cells of biological samples. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 Monocyte ATP6V1D expression distinguishes the efficiency of normal and cognitively impaired subjects.

[0015] Figure 2 Double-labeling immunofluorescence staining of NeuN (neuronal nuclei) and ATP6V1D in the cerebral cortex of normal people and AD patients.

[0016] Figure 3 The expression level of ATP6V1D in the brain tissue of AD patients and its correlation with the Mini-Scale Cognitive Examination (MMSE) score.

[0017] Figure 4 The specific molecular mechanism by which decreased ATP6V1D expression causes AD-like neuropathy and cognitive impairment.

[0018] Figure 5Overexpression of ATP6V1D improves AD-like neuropathy and cognitive impairment.

[0019] Figure 6 Ratio curve ranking of 153 drug compounds based on molecular docking analysis. DETAILED DESCRIPTION

[0020] Example 1

[0021] This example is used to illustrate that in the population database, ATP6V1D is downregulated in the brain tissue and peripheral blood of AD patients, and the expression of ATP6V1D is highly efficient in distinguishing normal and cognitively impaired populations. ATP6V1D can be used as a reference target for early diagnosis of AD.

[0022] Using multi-omics analysis techniques, Figure 1 A and D show that compared with the normal group, ATP6V1D was significantly reduced in monocytes of AD patients, and asterisks represent significant correlations: ***p<0.001, ****p<0.0001. In peripheral blood cells, it was found that the lysosomal ion channel-related protein ATP6V1D was significantly reduced in patients with mild cognitive impairment (MCI) and AD.

[0023] Logistic regression analysis was performed to plot the area under the curve to evaluate the efficiency of ATP6V1D in distinguishing normal from cognitively impaired populations. Figure 1 In BC and EF, the results of area under the ROC curve (AUC) analysis in multiple AD cohorts showed that changes in ATP6V1D in monocytes can effectively distinguish normal and cognitively impaired subjects: in the GSE63060 cohort, the AUC was 0.786, the sensitivity was 0.663, and the specificity was 0.750; in the GSE63061 cohort, the AUC was 0.663, the sensitivity was 0.641, and the specificity was 0.605.

[0024] Analysis of relevant data from GSE1297 and GSE28146 population databases revealed that Figure 3 A shows that compared with the normal group, ATP6V1D was significantly reduced in the brain tissues of patients in multiple AD cohorts. Figure 4 KM showed that ATP6V1D in AD patients continued to decrease with the course of the disease and was significantly correlated with MMSE.

[0025] By immunofluorescence co-staining ( Figure 2 ), we found that the expression of ATP6V1D in the cerebral cortex of AD patients was also significantly reduced.

[0026] The specific steps of immunofluorescence staining are as follows:

[0027] 1. Use 4% formaldehyde solution dissolved in PBS for tissue fixation at room temperature for 10-30 minutes.

[0028] 2. At room temperature, remove the fixative solution and wash with PBS (add 400 μL and shake gently) twice, 10 min each time.

[0029] 3. At room temperature, add 200 μL of 0.1% Triton X-100 solution dissolved in PBS for permeabilization for 5 min (to increase its permeability), and then aspirate the solution.

[0030] 4. Wash the tissue with PBS (add 400 μL and shake gently) twice at room temperature, 10 min each time.

[0031] 5. Take enough freshly prepared FITC-labeled phalloidin working solution to cover the tissue and incubate at room temperature in the dark for 30 minutes.

[0032] 6. Wash the tissue with PBS (add 400 μL and shake gently) 3 times, 5 min each time.

[0033] 7. Use fluorescent sealing agent containing DAPI to seal the slides.

[0034] 8. Under a laser confocal fluorescence microscope, select the FITC excitation / emission filter module (Ex / Em=193 / 517 nm) and the DAPI excitation / emission filter module (Ex / Em=364 / 454 nm) for corresponding observation and perform microscopic photography.

[0035] Example 2

[0036] This example is used to illustrate that decreased expression of ATP6V1D can cause AD-like neuropathy and cognitive impairment. Abnormal aggregation of Tau protein in AD may cause decreased expression of ATP6V1D, thereby causing lysosomal acidification disorders, which in turn lead to autophagy disorders and accumulation of Tau, thereby accelerating the progression of AD.

[0037] Analysis of relevant data from GSE1297 and GSE28146 population databases, Figure 4 GH showed that in human brain tissue, ATP6V1D expression was significantly negatively correlated with Tau pathological grade. Among them, Tau pathological grade includes neurofibrillary tangles (NTF) and Braak score. This suggests that abnormal aggregation of Tau protein may cause decreased ATP6V1D expression.

[0038] The hippocampal tissues of wild-type mice and P301L mice were taken, and the mRNA and protein expression levels of ATP6V1D were determined by RT-qPCR and WB experiments, respectively. RNA and protein were extracted from normal Hek293t cells and Hek293t cells transfected with Tau gene, and the above determination was also performed.

[0039] The specific experimental steps of RT-qPCR are as follows:

[0040] (1) Extraction of RNA:

[0041] 1. Add RNAisolater to fully cover the tissue surface, and then mix well by pipetting.

[0042] 2. Transfer the lysate to a 1.5 ml centrifuge tube, pipette repeatedly until fully lysed, and place on ice for 5 minutes.

[0043] 3. Add 1 / 5 volume of chloroform to the above lysate, shake vigorously for 15 seconds to form an emulsion, and let stand at 4℃ for 5 minutes.

[0044] 4.1 Centrifuge at 1200rpm (12000×g) at 4℃ for 15min. (This step must be centrifuged at low temperature, otherwise the product will have a small amount of genomic contamination.)

[0045] 5. Carefully remove the centrifuge tube. The solution is now divided into three layers: a colorless aqueous phase (upper layer), a white middle layer, and a red organic layer (lower layer). Carefully pipette the upper aqueous phase into a new centrifuge tube.

[0046] 6. Add an equal volume of pre-cooled isopropanol, mix by inverting, and let stand at 4°C for 10 min.

[0047] 7. Centrifuge at 11200 pm (12000×g) at 4°C for 10 min; a white precipitate can usually be seen.

[0048] 8. Carefully discard the supernatant, add 1 ml of 75% ethanol (RNase-free ddH2O), tap the bottom of the tube to suspend the precipitate, turn it upside down several times, and let it stand at room temperature for 3-5 minutes.

[0049] 9. Centrifuge at 11200 rpm (12000×g) at 4°C for 5 min and discard the supernatant.

[0050] 10. Dry the precipitate in an open air at room temperature in a clean environment for 2-5 minutes. Be careful not to over-dry it, otherwise it will make the RNA difficult to dissolve.

[0051] 11. Add an appropriate amount of RNase-free ddH2O to dissolve the precipitate. If necessary, gently blow it a few times with a pipette. After it is completely dissolved, take a small amount for testing and store the rest at -85 to -65°C.

[0052] 12. The product concentration can be detected by spectrophotometer, OD 260 / OD 280 A ratio between 1.8 and 2.2 indicates that the RNA purity is high. RNA concentration (ng / ul) = OD 260 × dilution factor × 40; or use Qubit direct test.

[0053] (2) Reverse transcription:

[0054] 1. Genomic DNA removal: Prepare the following mixture in an RNase-free centrifuge tube: 12ul RNase-free ddH2O, 4ul 4×gDNAwiper Mix, template RNA, and 1pg-1μg Total RNA. Mix by gently pipetting and incubate at 42℃ for 2min.

[0055] 2. Prepare the reverse transcription reaction system: Take 16ul of the reaction solution in the reaction tube in step 1, add 4ul of 5×HiScriptill qRT SuperMix, and mix gently by pipetting.

[0056] 3. Perform reverse transcription reaction: set the program in the PCR instrument to 37℃ for 15min and 85℃ for 5sec.

[0057] (3) qPCR:

[0058] 1. Prepare the following mixture in a qPCR tube: 10ul of 2×ChamQ SYBR gPCR Master Mix (Without ROX), 0.4ul of Primer1 (10uM), 0.4ul of Primer2 (10uM), and 8.2ul of Template DNA / cDNA.

[0059] 2. Perform qPCR reaction according to the following conditions

[0060]

[0061]

[0062] The specific steps of Western Blotting are as follows:

[0063] 1. Add an appropriate amount of strong RIPA lysis buffer (add weak RIPA lysis buffer to cells) to the hippocampal tissue of C57BL / 6J and P301L mice according to the tissue lysis buffer ratio (1:10), then ultrasonically disrupt and extract tissue protein (cells can be repeatedly frozen and thawed for lysis), centrifuge at 4°C, and collect the supernatant, and quantify the protein concentration using a BCA kit.

[0064] 2. After the quantification, add an appropriate amount of Loading buffer to the protein and cook at 95℃ for 10 minutes, then cool to room temperature.

[0065] 3. Prepare 10%-12% SDS-PAGE gel, take 20ug of boiled protein for loading, then perform 80V constant voltage electrophoresis for 30min to concentrate the protein, and then perform 120V constant voltage electrophoresis for 45min to separate the protein.

[0066] 4. Transfer the membrane. Soak the filter paper, sponge, methanol-activated PVDF membrane, and separation gel in electrotransfer buffer. Then clamp the membrane transfer clip in the order of white side, sponge, filter paper, NC membrane, separation gel, filter paper, sponge, and black side of the membrane transfer clip. Place the clip in the transfer instrument. Pour in the electrotransfer buffer and run at a constant current of 200 mA in an ice bath for 90 min.

[0067] 5. After the transfer, remove the NC membrane and block it with 5% skim milk powder (prepared with 1×TBST) at room temperature for 1 hour. Then wash the NC membrane slightly, cut the corresponding bands according to the molecular weight of the target protein, and finally put the bands into the diluted primary antibody solution (ATP6V1D, Tau404) and incubate them at 4°C with shaking overnight.

[0068] 6. After incubation with the primary antibody, wash the strips three times with 1×TBST, 10 min each time, then add an appropriate amount of diluted secondary antibody (goat anti-mouse or goat anti-rabbit) and incubate on a shaker at room temperature for 1 h.

[0069] 7. After incubation with the secondary antibody, wash the strips three times with 1×PBST for 10 min each time, then mix equal volumes of eECL-A and eECL-B (Thermo, 34075) in a 1:1 ratio, drip evenly onto the target strips in a dark environment, and use a chemiluminescence imager for exposure and imaging.

[0070] The V-ATPase level in hippocampal tissue specimens of P301L mice was determined using a mouse V-ATPase ELISA kit (double antibody sandwich method). The microplate was coated with purified mouse V-ATPase antibody to prepare a solid-phase antibody, and V-ATPase was added to the microwells coated with monoclonal antibodies in sequence, and then combined with HRP-labeled V-ATPase antibody to form an antibody-antigen-enzyme-labeled antibody complex. After thorough washing, the substrate TMB was added for color development. TMB was converted into blue under the catalysis of HRP enzyme, and converted into the final yellow under the action of acid. The depth of color was positively correlated with the V-ATPase in the sample. The absorbance (OD value) was measured at a wavelength of 450nm using an enzyme reader, and the concentration of mouse V-ATPase in the sample was calculated using a standard curve.

[0071] HEK293t cell lines transfected with ATP6V1D plasmid, HEK293t cells treated with tau, and HEK293t cells treated with tau and transfected with ATP6V1D plasmid, and cell samples transfected with mCherry-GFP-LC3 (when the autophagosome reaches the lysosome, GFP is quenched in an acidic environment and only mCherry emits red light) were collected for autophagy flux detection.

[0072] The specific steps of transfection are as follows:

[0073] 1. Cultivate HEK293t cells: One day before transfection, inoculate 0.5-2×105 HEK293t cells in 500μ1 antibiotic-free culture medium to ensure that the turbidity of the cells reaches 90%-95% during transfection.

[0074] 2. Transfection of target gene:

[0075] For each transfection sample, prepare as follows:

[0076] a. Dilute DNA in 250 μl serum-free Opti-MEMI Low Serum Medium (or other serum-free medium) and mix gently.

[0077] b. Gently mix LipofectamineTM 2000 before use, then dilute the corresponding amount in Opti-MEM medium and let it stand at room temperature for 5 minutes. Mix LipofectamineTM 2000 and DNA dilution within 30 minutes.

[0078] c. After standing at room temperature for 5 minutes, mix LipofectamineTM2000 and DNA dilution (total volume is 500u1). Mix gently and stand at room temperature for 20 minutes (the mixture may look turbid). The mixture will not lose its effectiveness within 6 hours at room temperature.

[0079] 2. Prepare a six-well cell culture plate, add 500ul of the mixture to each well of the cell plate, and gently shake the cell plate back and forth to mix the mixture with the culture medium in the well.

[0080] 3. Before testing the expression of the target gene, the cells are cultured in an incubator at 37°C for 18-48 hours. The culture medium does not need to be changed at this time, but it needs to be replaced after 4-6 hours.

[0081] Example 3

[0082] This example is used to illustrate that overexpression of ATP6V1D has an improving effect on AD-like neuropathy and cognitive impairment, and can be used as a therapeutic target for AD.

[0083] The cell samples of Example 2 were taken for WB experiment, RT-qPCR experiment and autophagy fluorescence flux detection to analyze the improvement of cellular Tau pathology and autophagy flux after overexpression of ATP6V1D.

[0084] Figure 4 In L, compared with the Control group and the Tau group, after overexpression of ATP6V1D, GFP can emit weak green fluorescence under a fluorescence microscope, indicating that the acidic environment of the lysosome has been restored compared with the Tau group, and mCherry emits red fluorescence, and the two are superimposed to form a weak yellow fluorescence; Figure 4 M, compared with the Control group and Tau group, the number of lysosomes increased after overexpression of ATP6V1D compared with the Tau group, indicating that overexpression of ATP6V1D can improve the lysosomal acidification disorder of Tau-treated HEK-293t cells.

[0085] At the same time, PCR experiments showed that ( Figure 5 A), compared with the OE-Tau group (AD model cells), the overexpression of ATP6V1D in the OE-Tau+ATP6V1D group significantly reversed the ATP6V1D expression defect caused by Tau; WB experiments showed ( Figure 5 BC), the expression of Tau5 protein was significantly decreased in the OE-ATP6V1D group, indicating that overexpression of ATP6V1D could significantly alleviate Tau pathology.

[0086] Example 4

[0087] This example is used to illustrate the preparation method of a kit for detecting Alzheimer's disease. The kit is used to detect the content of ATP6V1D protein in peripheral blood cells, and mainly includes the following reagents:

[0088] Cell Lysis Buffer: Lyses blood cells and releases intracellular proteins.

[0089] Formula: 50 mM Tris-HCl, 150 mM NaCl, 1% Triton X-100, pH 7.4; contains protease inhibitors.

[0090] ATP6V1D capture antibody: coated on a 96-well plate to capture ATP6V1D protein in the sample.

[0091] Detection Antibody: Secondary antibody that binds to ATP6V1D protein and is labeled with an enzyme or fluorescent marker.

[0092] Enzyme substrate solution: used for color development reaction, such as TMB substrate (for HRP-labeled antibodies).

[0093] Washing buffer: PBS (pH 7.4) + 0.05% Tween-20.

[0094] Standard: Purified ATP6V1D protein, for standard curve.

[0095] Blocking solution: such as PBS buffer containing 1% BSA, used to reduce nonspecific binding.

[0096] The preparation steps of the kit are as follows:

[0097] 1. Capture antibody coating

[0098] Materials: ATP6V1D capture antibody, 96-well plate.

[0099] Prepare capture antibody solution: dilute the antibody to 1-10 μg / mL (optimal concentration determined based on antibody affinity and preliminary experiments) and dissolve in carbonate buffer (0.05 M, pH 9.6).

[0100] Add antibody: Add 100 μL of capture antibody solution to each well and incubate overnight at 4°C.

[0101] Blocking: The next day, discard the antibody solution, add 200 μL of blocking solution (such as 1% BSA in PBS) to each well, and block at 37°C for 1 hour.

[0102] Wash three times: Wash the plate three times with wash buffer (PBS + 0.05% Tween-20), leaving it for 1 minute each time.

[0103] 2. Detection Antibody Labeling and Validation

[0104] Labeling: ATP6V1D detection antibody can be coupled to HRP enzyme or labeled with other fluorescent dyes such as FITC.

[0105] Verification: Use Western blot or ELISA to verify the recognition ability and labeling efficiency of the labeled antibody for ATP6V1D.

[0106] 3. Preparation of Standards

[0107] Prepare a series of dilutions of purified ATP6V1D protein at concentrations of 0, 1, 5, 10, and 20 ng / mL for use in drawing a standard curve. Dilute the standard with cell lysis buffer to approximate the background of the actual sample.

[0108] 4. Assembling the kit

[0109] The capture antibody-coated 96-well plate, detection antibody, wash buffer, TMB substrate solution, blocking solution, and ATP6V1D standard were placed in separate containers, labeled, and assembled into a kit and stored at 2-8°C.

[0110] Instructions for use of the kit:

[0111] 1. Sample processing

[0112] Peripheral blood samples were collected and peripheral blood mononuclear cells (PBMCs) were isolated by density gradient centrifugation. PBMCs were resuspended in cell lysis buffer and incubated on ice for 30 minutes with intermittent gentle shaking. After full lysis, the cells were centrifuged at 4°C, 12,000 rpm for 10 minutes, and the supernatant was collected for detection.

[0113] 2. Detection steps

[0114] Sample loading: Add 100 μL of sample lysate or standard to each well of a 96-well plate coated with capture antibody and incubate at room temperature for 1 hour.

[0115] Washing: Add wash buffer to each well and wash the plate five times, leaving it alone for 1 minute each time.

[0116] Add detection antibody: Add 100 μL of enzyme-labeled detection antibody solution to each well and incubate at room temperature for 30 minutes.

[0117] Wash: Wash five times with wash buffer.

[0118] Color development: Add 100 μL of TMB substrate solution and incubate in the dark for 10-15 minutes until the color develops.

[0119] Termination: Add 50 μL 2M sulfuric acid to terminate the reaction and read the OD value at a wavelength of 450 nm using a microplate reader.

[0120] 3. Data Analysis

[0121] The OD value of the sample was converted into ATP6V1D concentration using the standard curve to assess the ATP6V1D content in peripheral blood cells.

[0122] Example 5

[0123] This example is used to illustrate that there is a drug, Isoliquiritin apioside, which can increase the activity of V-ATPase (v-ATPase), thereby increasing the expression level of ATP6V1D. This drug screening method is based on the systematic screening and molecular verification of 153 candidate drugs, and finally determines that Isoliquiritin apioside is an ideal drug.

[0124] 1. Drug Screening Methods

[0125] Compound library construction: 153 candidate drug compounds were selected to construct a drug compound library.

[0126] Determination of the optimal concentration: By consulting relevant literature, the optimal concentration data of each drug in HEK293t cells were obtained to ensure that the dosage concentration had a physiological effect and did not affect cell activity.

[0127] Cell model construction: A stably transfected MAPT (microtubule-associated protein tau gene) cell line was established in HEK293t cells as an experimental model to simulate the cellular environment of neurodegenerative diseases.

[0128] Drug treatment: In the HEK293t-MAPT stable cell line, each drug was uniformly treated at the optimal concentration. After adding each drug, the cells were cultured for another 24 hours.

[0129] Supernatant collection: After 24 hours, the cell culture supernatant of each experimental group was collected and stored at 4°C for subsequent detection.

[0130] 2.ELISA kit detection

[0131] The v-ATPase content in the supernatant of each group of samples was quantitatively determined using an ELISA kit for v-ATPase. The ratio of the v-ATPase content in each drug group to that in the control group was calculated based on the ELISA test results, and a ratio curve was drawn to show the effects of different drugs on v-ATPase expression.

[0132] 3. Molecular Docking Analysis

[0133] Molecular docking analysis was performed on the selected candidate drugs to calculate the binding activation energy between the drug and v-ATPase and predict the potential mechanism of action of the drug on v-ATPase. The effectiveness and affinity of each drug were comprehensively evaluated based on the ratio curve ranking and the activation energy data of molecular docking.

[0134] 4. Determine the best drug

[0135] Among all the candidate drugs, Isoliquiritin apioside showed the best effect ( Figure 6 ), whose molecular formula is as follows:

[0136]

[0137] Specifically, it has a higher v-ATPase expression ratio in the ELISA kit test and a lower molecular docking activation energy, showing good binding ability and action potential.

[0138] 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 ATP6V1D 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 ATP6V1D is shown in SEQ ID NO.

1.

3. The use according to claim 1, characterized in that The ATP6V1D 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: Includes reagents for detecting the amount of ATP6V1D 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 ATP6V1D 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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