Use of ILDR3 gene and / or protein in preparation of drugs for preventing and / or treating tau-related diseases, diagnostic products

Abstract

The application belongs to the technical field of biological medicine, and provides application of ILDR3 gene and / or protein in preparation of a drug for preventing and / or treating a Tau-related disease and a diagnostic product. The ILDR3 provided in the application is a key treatment target of the Tau-related disease, can simultaneously intervene in multiple core links of the Tau pathology, including promoting degradation of the Tau protein, inhibiting excessive phosphorylation thereof and blocking abnormal aggregation thereof. Experimental data show that the expression of ILDR3 is down-regulated in the Tau-related disease, knocking out ILDR3 aggravates the Tau pathology and cognitive impairment, and overexpression of ILDR3 significantly improves learning and memory function of a Tau pathology model and reduces nerve damage. Therefore, the ILDR3 gene and / or protein can be applied to preparation of a drug for preventing and / or treating a Tau-related disease and a diagnostic product.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and particularly relates to the use of ILDR3 genes and / or proteins in the preparation of drugs and diagnostic products for preventing and / or treating Tau-related diseases. Background Art

[0002] Tauopathies are a class of neurodegenerative diseases characterized by abnormal aggregation of the tau protein, including Alzheimer's disease, amyotrophic lateral sclerosis, and Parkinson's disease. Currently, there is no cure for tauopathies, and existing treatments only partially alleviate symptoms. Therefore, research on drugs targeting the pathological mechanisms of tauopathies and their progression has become a hot topic. Tau-targeted immunotherapies, such as monoclonal antibodies (e.g., E2814), antisense oligonucleotides (e.g., MAPTRx), tau aggregation inhibitors (e.g., TRx0237), and small molecule inhibitors (e.g., LY3372689 and TPI-287), have entered clinical trials, but most have been discontinued due to insufficient efficacy or side effects, highlighting the complexity and challenges of treating tauopathies. Therefore, the development of novel therapeutic targets and drugs with innovative mechanisms of action to more effectively intervene in or even reverse tauopathology has become a critical scientific challenge and a significant clinical need in this field. Summary of the Invention

[0003] To solve the above technical problems, the present invention provides the use of ILDR3 (Immunoglobulin-Like Domain Containing Receptor 3) gene and / or ILDR3 protein as targets in the preparation of drugs for preventing and / or treating Tau-related diseases and products for diagnosing Tau-related diseases.

[0004] The first aspect of the present invention is to provide the use of ILDR3 gene and / or ILDR3 protein as a target in the preparation of a drug for preventing and / or treating Tau-related diseases.

[0005] The ILDR3 gene and / or ILDR3 protein described in the present invention are ubiquitous in humans and animals and have cross-species properties. Therefore, the application of the ILDR3 gene and / or ILDR3 protein described above in the present invention is not limited to humans, but also includes other animal species containing the ILDR3 gene and / or ILDR3 protein, such as cats, dogs, monkeys, mice, etc.

[0006] The second aspect of the present invention is to provide a substance capable of promoting the expression and / or activity of the ILDR3 gene and / or protein, including an expression vector containing an ILDR3 encoding gene and / or a substance that targets and regulates the expression and / or activity of the ILDR3 protein by regulating the signaling pathway and / or a specific regulatory substance for the upstream and downstream signaling pathways of the ILDR3 protein itself.

[0007] An ILDR3 expression and / or activity promoter refers to a substance that can promote the expression and / or activity of the ILDR3 gene and / or protein. Any substance that acts through ILDR3 using any means or method falls within the scope of protection of the present invention. Preferably, the ILDR3 gene and / or ILDR3 protein expression promoter includes: a recombinant expression vector containing the ILDR3 gene or a gene fragment with greater than 90% homology to the ILDR3 gene; or at least one of a polypeptide, protein, nucleic acid, nucleic acid aptamer, polysaccharide, natural active substance, or biological agent that can upregulate the expression of the ILDR3 gene and / or ILDR3 protein.

[0008] Among them, the recombinant expression vector includes any one of a vector-based eukaryotic expression plasmid, adenovirus, adeno-associated virus, lentivirus, retrovirus, LNP liposome, microinjection technology, gene editing system elements, and homologous recombination vector that specifically targets the ILDR3 gene and / or ILDR3 protein.

[0009] The expression vector having the ILDR3 encoding gene is selected from any one of a peptide specifically targeting ILDR3, a small molecule, an agonist of the ILDR3 protein, a compound, a combination drug, adenovirus, adeno-associated virus, lentivirus, LNP liposome, a homologous recombination vector, microinjection technology, a product containing gene editing system components, and a nucleic acid drug.

[0010] As further preferred, the ILDR3 gene and / or ILDR3 protein expression promoter targets and regulates the expression of the ILDR3 gene and / or ILDR3 protein by regulating signal pathways.

[0011] The third aspect of the present invention provides the use of supplementing ILDR3 protein, products containing ILDR3 amino acid sequences, and cells expressing ILDR3 with ILDR3 as a therapeutic target in the preparation of drugs for preventing and / or treating Tau-related diseases.

[0012] Preferably, the cells expressing ILDR3 with ILDR3 as the target are selected from any one or more of glial cells, neurons, endothelial cells, and stem cells.

[0013] Preferably, the stem cells are selected from any one or more of mesenchymal stem cells derived from bone marrow, fat, umbilical cord, induced pluripotent stem cells, and embryonic stem cells.

[0014] Use of the aforementioned ILDR3 expression and / or activity promoter, ILDR3 protein, product comprising ILDR3 amino acid sequence, or ILDR3-expressing cell with ILDR3 as a therapeutic target in any one or more of the following:

[0015] (1) Products for the preparation of drugs for the prevention and / or treatment of Tau-related diseases;

[0016] (2) Prepare products for improving the ability of neurons and glial cells to process Tau;

[0017] (3) Prepare products for maintaining the ability of neurons and glial cells to process Tau;

[0018] (4) Application in the preparation of products for promoting Tau protein degradation;

[0019] (5) Application in the preparation of products for inhibiting Tau protein phosphorylation;

[0020] (6) Application in the preparation of products for inhibiting Tau protein aggregation;

[0021] (7) Preparation of products for the prevention and / or treatment of Aβ-related diseases.

[0022] Preferably, the ILDR3 gene and / or ILDR3 protein expression promoter is a substance that can enhance the expression and / or activity and / or function of the ILDR3 gene and / or ILDR3 protein.

[0023] The above-mentioned Tau-related diseases are selected from any one or more of Alzheimer's disease, amyotrophic lateral sclerosis, Parkinson's disease, dementia pugilistica, Down syndrome, traumatic brain injury, Guam amyotrophic lateral sclerosis / Parkinson's syndrome-dementia complex, non-Guam motor neuron disease with neurofibrillary tangles, argyrophilic grain dementia, corticobasal degeneration, diffuse neurofibrillary tangles with calcifications, frontotemporal dementia, chromosome 17-related frontotemporal dementia with Parkinson's syndrome, Hallevorden-Spatz disease, Niemann-Pick disease type C, globus pallidus-ponto-nigral degeneration, Pick's disease, progressive subcortical gliosis, progressive supranuclear palsy, tangled dementia, postencephalitic Parkinson's syndrome and myotonic dystrophy.

[0024] A fourth aspect of the present invention provides a drug comprising at least one of the aforementioned ILDR3 gene, ILDR3 protein, and ILDR3 gene and / or ILDR3 protein expression promoters, as a drug composition and / or auxiliary drug composition for preventing and / or treating Tau-related diseases, and using the drug as the main functional ingredient of the drug.

[0025] Wherein, the pharmaceutical composition is selected from any one of tablets, capsules, powders, pills, granules, solutions, suspensions, syrups, injections, suppositories, inhalants, and sprays;

[0026] The auxiliary material is selected from any one or more of diluents, excipients, fillers, binders, wetting agents, disintegrants, absorption promoters, surfactants, adsorption carriers, and lubricants.

[0027] As a further preferred embodiment, the content of the functional ingredient in the drug is 1%-99%, that is, the functional ingredient added to the drug (at least one of the ILDR3 gene, ILDR3 protein, and an expression promoter of the ILDR3 gene and / or ILDR3 protein) accounts for 1%-99% of the total weight of the drug.

[0028] A fifth aspect of the present invention provides the use of a reagent for detecting the expression of the ILDR3 gene and / or ILDR3 protein in the preparation of a product for diagnosing Tau-related diseases, wherein the product comprises any one of a chip, a diagnostic reagent, and a diagnostic kit;

[0029] Preferably, the product contains antibodies that specifically bind to the ILDR3 protein and / or peptide fragments of the ILDR3 protein, and / or contains primers that specifically amplify the ILDR3 gene, and / or contains a probe that specifically detects the ILDR3 gene;

[0030] Preferably, the test sample for the drug-assisted diagnosis product for Tau-related diseases is selected from any one or more of tissue, cerebrospinal fluid, whole blood, serum, plasma, hair, and excrement;

[0031] Preferably, the products include chips, diagnostic reagents, and diagnostic kits.

[0032] Preferably, the product is used to detect the expression of the ILDR3 gene and / or ILDR3 protein. When the expression of the ILDR3 gene or ILDR3 protein is downregulated, a Tau-related disease is diagnosed.

[0033] The present invention has the following advantages and effects compared to the prior art:

[0034] (1) This invention provides for the first time a therapeutic strategy targeting ILDR3 to regulate multiple Tau pathological processes in Tau-related diseases by single-target regulation of ILDR3 expression and / or activity, and confirms for the first time the following:

[0035] (a) ILDR3 is involved in the degradation of pathological Tau in microglia;

[0036] (b) ILDR3 is involved in the degradation of pathological Tau in astrocytes;

[0037] (c) Inhibitory effect of ILDR3 on pathological Tau protein phosphorylation;

[0038] (d) ILDR3 significantly inhibits the aggregation of pathological Tau protein;

[0039] (2) The present invention is the first to demonstrate that targeting ILDR3 and overexpressing ILDR3 via adeno-associated virus has important beneficial effects in the treatment of Tau-related diseases such as Alzheimer's disease: significantly improving learning and memory functions, reducing neuronal damage, and reducing the levels of Tau protein and its phosphorylated protein;

[0040] (3) The present invention further provides a reagent for detecting the expression of ILDR3 gene and / or ILDR3 protein in Alzheimer's disease mice and Tau disease mouse models (PS19 mice) based on the expression changes of ILDR3 protein, and uses it in the preparation of products for diagnosing Tau-related diseases. That is, the expression of ILDR3 gene and / or ILDR3 protein is used as a biomolecular marker for the detection of Tau-related disease diagnostic products, and the expression of ILDR3 gene and / or ILDR3 protein is detected by the product. When the expression of ILDR3 gene and / or ILDR3 protein is downregulated, Tau-related disease is diagnosed. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 The results show that ILDR3 expression is decreased in the brain tissues of mice with Tau-related diseases and Alzheimer's disease, where A is the Western blot result of ILDR3 in Tau-related diseases, and B is the Western blot result of ILDR3 expression decreased in the brain tissues of mice with Alzheimer's disease;

[0042] Figure 2 Figure 3 shows the results of overexpression of ILDR3 promoting the degradation of P301L Tau, where A is the Western blot result of the effect of ILDR3 on the degradation of P301L Tau, and B is the grayscale value quantitative result of A;

[0043] Figure 3The results of overexpression of ILDR3 inhibiting P301L Tau phosphorylation, where A is the Western blot result of ILDR3 on P301L Tau phosphorylation, and B is the gray value quantitative result of A;

[0044] Figure 4 The results of overexpression of ILDR3 inhibiting the aggregation of P301L Tau, where A shows the aggregation degree of P301LTau by ILDR3, and B shows the Western blot results of P301L Tau in the control group and ILDR3 overexpression group;

[0045] Figure 5 Overexpression of ILDR3 improves the ability of microglia to process P301L Tau, where A is the Western blot detection result of Tau levels in cells and supernatant, and B is the gray value quantification result of A;

[0046] Figure 6 Overexpression of ILDR3 improves the ability of astrocytes to process P301L Tau. A is the Western blot detection result of Tau levels in cells and supernatant, and B is the gray value quantification result of A.

[0047] Figure 7 Figures 1 and 2 show that ILDR3 gene knockout leads to aggravated Tau pathology, where A shows the water maze latency, B shows the novel object recognition results, C shows the ELISA results for Tau levels in cerebrospinal fluid, D shows the results for NP-40-soluble and SDS-soluble Tau and phosphorylated Tau in hippocampal tissue, and E shows the immunofluorescence staining results for the neuronal marker NeuN and the astrocyte activation marker GFAP.

[0048] Figure 8 To overexpress ILDR3 to prevent or delay the progression of Tau pathology, A shows the animal experimental model, B shows the water maze latency results, C shows the Y maze spontaneous alternation percentage results, D shows the ELISA test results for Tau levels in cerebrospinal fluid, E shows the PET / CT results, F shows the results of NP-40-soluble and SDS-soluble Tau and phosphorylated Tau in hippocampal tissue, and G shows the immunofluorescence staining results for the neuronal marker NeuN and the astrocyte activation marker GFAP.

[0049] Figure 9Overexpression of ILDR3 ameliorates neuronal damage caused by PFF Tau. (A) Shows the animal experimental model; (B) Water maze latency results; (C) Y-maze spontaneous alternation percentage results; (D) Cerebral spinal fluid Tau ELISA test results; (E) NP-40-soluble and SDS-soluble Tau and phosphorylated Tau in hippocampal tissue; (F) Immunofluorescence staining results for Tau phosphorylated protein AT8 and neuronal marker NeuN. DETAILED DESCRIPTION

[0050] In order to enable those skilled in the art to better understand the present invention, the present invention will be further described in conjunction with specific embodiments.

[0051] Test Example 1

[0052] 1. Western Blot analysis of ILDR3 expression in hippocampal tissue of Tau-related disease model mice (PS19 P301S mice), 20-month-old mice, and 4-month-old wild-type C57BL / 6 mice. The specific experimental methods are as follows:

[0053] ① Total protein extraction: Add 2 mg / 200 μL RIPA lysis buffer (containing protease inhibitors) to the hippocampus tissue and homogenize. Lyse on ice for 30 minutes. Place the sample symmetrically in a centrifuge and centrifuge at 4°C, 12,000 rpm, for 10 minutes. Transfer the supernatant to a new EP tube.

[0054] ② Protein sample preparation: Detect protein concentration using a BCA assay kit; add 5× SDS-Loading Buffer to the protein sample, boil the sample at 100°C for 10 minutes, and centrifuge briefly;

[0055] ③ Protein electrophoresis: Select 4-20% gradient gel, add 20 μg protein to each well, 120V, 80min;

[0056] ④ Transfer: Remove the protein gel and place the gel and 0.2μm NC membrane between filter paper soaked in buffer to make a transfer "sandwich". Place it in the transfer tank and add NCM rapid transfer buffer. Transfer at 200mA for 2h.

[0057] ⑤ Blocking: Remove the transferred membrane, add an appropriate amount of 5% skim milk powder, and shake slowly at room temperature for 1 hour. Wash with TBST three times, 10 minutes each time;

[0058] ⑥ Primary antibody incubation: dilute ILDR3 primary antibody at a dilution of 1:1000 in blocking buffer; dilute β-actin primary antibody at a dilution of 1:10000 in blocking buffer. Add the diluted primary antibody to the antibody incubation, evenly cover the membrane, shake at low speed at 4°C overnight, and wash three times with TBST, each time for 10 minutes.

[0059] ⑦ Secondary antibody incubation: Dilute the secondary antibody with TBST, add it to the antibody incubation, evenly cover the membrane, and incubate at room temperature with shaking for 1 hour. After incubation, aspirate the secondary antibody, add TBST, and wash three times, each time for 10 minutes;

[0060] ⑧ Development: Mix the luminescent solutions (Solution A and Solution B) in a 1:1 ratio (keep away from light). Use a pipette to draw an appropriate amount of luminescent solution to cover the PVDF membrane. Expose and collect images on an ECL luminometer.

[0061] 2. Data Analysis and Results

[0062] Image J software was used to calculate the gray value of each band, and Prism software was used to statistically analyze the data of three independent repeated experiments. P Statistical significance was observed when the value was <0.05. Western blot images were assembled and sorted using Adobe Illustrator.

[0063] Figure 1 Compared with wild-type mice of the same age, ILDR3 expression in the hippocampus of Tau-related disease model mice was significantly decreased (e.g. Figure 1 Compared with young mice, ILDR3 expression in the hippocampus of Alzheimer's mice was significantly decreased (as shown in Figure 2A). Figure 1 (shown in B).

[0064] From the above, it can be seen that the expression level of ILDR3 protein is related to the occurrence of Tau-related diseases. The expression level of ILDR3 gene protein is significantly downregulated in Tau-related diseases. Therefore, it can be used as a molecular marker for Tau-related diseases and used for clinical auxiliary diagnosis of Tau-related diseases.

[0065] Experimental Example 2: ILDR3 overexpression promotes Tau protein degradation

[0066] 1. Cell culture and treatment

[0067] HEK293 cells were routinely cultured in DMEM medium supplemented with 10% FBS, 1% penicillin, and streptomycin in a 37°C, 5% CO2 incubator. The medium was changed every two days and the cells were passaged at a 1:3 ratio.

[0068] P301L Tau is a mutant of Tau protein. In the pathological process of Tau, mutant Tau protein plays an important role in the pathological progression of Tau-related diseases. Therefore, we used mutant P301L Tau protein for subsequent research. For cell treatment, 1.4 μg of P301L Tau plasmid and 0.6 μg of human ILDR3 (NM_205834.4) or its control plasmid were transfected into the cells using PEI reagent. The transfection method was carried out according to the PEI transfection instructions. 36 hours after transfection, the cells were treated with protein synthesis inhibitor - cycloheximide (CHX) for different time periods (such as Figure 2 shown).

[0069] 2. Experimental methods

[0070] (1) Western Blot detection of Tau expression in HEK293 cells. The specific experimental methods are as follows:

[0071] ① Total cell protein extraction: HEK293 cells were digested and plated in 6-well cell culture plates (n = 3). After cells adhered, they were harvested into 1.5 mL EP tubes and washed twice with PBS. 100 μL of RIPA lysis buffer (containing protein synthesis inhibitors) was added to each well and lysed on ice for 30 min. The samples were centrifuged symmetrically at 12,000 rpm at 4°C for 10 min. The supernatant was transferred to a fresh EP tube.

[0072] ②The Western Blot experimental steps were the same as those described in Experimental Example 1.

[0073] Experimental results: Figure 2 As shown in the results, ILDR3 overexpression can significantly accelerate the degradation of Tau, indicating that ILDR3 protein has a promoting effect on the degradation of Tau.

[0074] Experimental Example 3: ILDR3 overexpression inhibits Tau phosphorylation

[0075] 1. Experimental methods

[0076] (1) HEK293 cells were transfected with different amounts of P301L Tau plasmid and human ILDR3 (NM_205834.4) or its control plasmid using PEI reagent. The expression levels of Tau in the control group and the ILDR3 overexpression group were consistent at the time of final sample collection. 30 hours after transfection, the cells were treated with phosphatase inhibitor (OA) for 6 hours, and then the total cell protein was collected.

[0077] Wash twice with PBS, add 100 μL of RIPA lysis buffer (containing protease inhibitors) to each well, and lyse on ice for 30 minutes. Place the samples symmetrically in a centrifuge and centrifuge at 12,000 rpm for 10 minutes at 4°C. Transfer the supernatant to a new EP tube.

[0078] (2) The Western Blot experimental steps were the same as those described in Experimental Example 1. The primary antibodies used were AT8 and Ser396, which target phosphorylated Tau, to detect the phosphorylation level of Tau.

[0079] Table 1 ILDR3 promotes Tau degradation

[0080]

[0081] Experimental results: Figure 3 As shown in the Figure 3, compared with the control group, ILDR3 overexpression significantly reduced the level of phosphorylated Tau AT8, confirming that ILDR3 has an inhibitory effect on Tau phosphorylation.

[0082] These results indicate that ILDR3 has an inhibitory effect on the phosphorylation of Tau protein.

[0083] Experimental Example 4: ILDR3 Overexpression Inhibits Tau Aggregation

[0084] (1) P301L Tau was constructed on fluorescent complementation vectors (pBiFC-VN55 (l152) and pBiFC-VC155). Bimolecular fluorescence complementation technology was used to study the interaction between P301L proteins. The principle is to cut Venus into two complementary but non-luminescent fragments (such as N-terminal and C-terminal fragments), and express them in fusion with P301L Tau respectively. Fluorescent signals were generated under a microscope, which visually reflected the interaction between P301L Tau.

[0085] (2) Cells were transfected with P301L Tau plasmids (pBiFC-VN55 (l152) and pBiFC-VC155) and human ILDR3 (NM_205834.4) or its control plasmid using PEI reagent. The amount of P301L Tau plasmids (pBiFC-VN55 (l152) and pBiFC-VC155) in the same treatment well was consistent. The amount of plasmid transfection in the control group and the ILDR3 group was different, but the Tau expression levels in the control group and the ILDR3 overexpression group were consistent when the samples were finally collected. Three replicates were set up for each group.

[0086] (3) 36 hours after transfection, the cells were observed and scanned using a fluorescence microscope under 488 emission light. Protein was also extracted and quantified.

[0087] Experimental results: Figure 4As shown, in HEK293 cells, the interaction between P301L and Tau proteins produced green fluorescence. Compared with the control group, the ILDR3 overexpression group significantly reduced the green fluorescence level while maintaining the same Tau protein expression level as the control group. This confirms that ILDR3 overexpression can inhibit the interaction between P301L and Tau proteins and reduce their aggregation.

[0088] This shows that ILDR3 protein can inhibit the aggregation of Tau proteins.

[0089] Experimental Example 5: ILDR3 Overexpression Improves the Tau Processing Ability of Microglia and Astrocytes

[0090] 1. Cell selection and culture

[0091] Human microglial cell lines HMC3 and glial cell lines H4 were cultured. H4 cells were routinely cultured in DMEM / F12 medium supplemented with 10% FBS, 1% penicillin, and streptomycin, while HMC3 cells were routinely cultured in DMEM / F12 medium supplemented with 10% FBS, 1% penicillin, and streptomycin in a 37°C, 5% CO2 incubator. Cells were cultured with medium replacement every two days and passaged at a 1:3 ratio.

[0092] 2. Construction of ILDR3 overexpression vector

[0093] (1) Obtain the human ILDR3 (NM_205834.4) gene sequence from NCBI;

[0094] (2) The ILDR3 gene sequence was synthesized by Sangon Biosynthesis Co., Ltd. and constructed into the mammalian cell overexpression plasmid PQCXIP.

[0095] 3. Construction of ILDR3-overexpressing HMC3 and glial cell lines and H4 cells

[0096] (1) Inoculate 293T cells into a 15 cm dish and culture overnight until the cell density reaches approximately 70-80% on the next day;

[0097] (2) Before transfection, replace the culture medium in the culture dish with 20 mL of fresh culture medium;

[0098] (3) Configure the overexpression lentiviral packaging system in order. For one 15cm culture dish to be transfected, the following transfection reagents need to be configured:

[0099] Solution A: 500 μL DMEM medium, 10 μg PQCXIP-ILDR3 overexpression plasmid / control plasmid, 10 μg packaging plasmid pMD1g / pRRE; 10 μg packaging plasmid pRSV-Rev and 10 μg envelope plasmid pMD2.G;

[0100] Solution B: 500 μL DMEM medium, 120 μL PEI transfection reagent. After leaving Solution A and Solution B at room temperature for 5 minutes, add Solution B and mix with Solution A. Let it sit at room temperature for 20 minutes, then evenly distribute to the corresponding culture dish.

[0101] (4) After culturing in a 37°C, 5% CO2 incubator for 12 hours, replace with fresh culture medium;

[0102] (5) After continuing to culture in the incubator for 48 hours, collect the culture supernatant and concentrate the virus by centrifugation.

[0103] 4. Lentiviral infection of HMC3 cells

[0104] a. Seed appropriate amounts of HMC3, glial cell lines, and H4 cells into 6-well plates and culture overnight so that the cell density reaches approximately 70-80% on the next day.

[0105] b. Discard the old culture medium and add 2 mL of fresh culture medium and 0.1 mL of the ILDR3 overexpression lentivirus packaged in the previous step. Gently mix thoroughly and incubate at 37°C, 5% CO2 for 12 hours.

[0106] c. Discard the old culture medium, add 2 mL of fresh culture medium, and incubate at 37°C, 5% CO2 for 36 hours.

[0107] d. Add 1 μg / mL puromycin to select positive cells. After approximately 2 to 3 days, all uninfected cells will be killed, leaving only positive cells, which are ILDR3-overexpressing cells or control cells.

[0108] 5. Detection of overexpression

[0109] The above-mentioned experimental method was used to extract and detect the expression level of ILDR3 protein in the screened cells. The specific steps were the same as above.

[0110] The expression levels of ILDR3 protein were significantly increased in HMC3, glial cell line and H4 cells ( Figure 5 and Figure 6 );

[0111] In summary, Figure 5-6 It can be seen that ILDR3 overexpressing cell lines were successfully constructed in both cell lines.

[0112] 6. Cell Processing

[0113] 1 μg / mL P301L Tau protein was cultured in HMC3, glial cell lines, and H4 cells for 24 hours, and then the residual Tau protein levels in the cells and culture medium of HMC3, glial cell lines, and H4 cells were detected.

[0114] 7. Western Blot detection of Tau residues in two cells

[0115] Experimental results:

[0116] like Figure 5 and Figure 6 As shown, in HMC3, glial cell lines, and H4 cells, the residual Tau levels in the culture supernatant and total intracellular protein of ILDR3 (NM_205834.4)-overexpressing cells were lower than those in the control group, demonstrating that ILDR3 overexpression can increase the ability of HMC3 and glial cell line H4 cells to process P301L Tau protein.

[0117] This indicates that ILDR3 can enhance the Tau processing ability of microglia and glial cells.

[0118] Experimental Example 6 ILDR3 gene knockout causes aggravated Tau pathology, Aβ accumulation, and learning and cognitive decline in mice

[0119] 1. Western Blot assay to detect Tau pathology.

[0120] (1) Extraction of mouse hippocampal tissue protein

[0121] Hippocampal tissue was lysed on ice for 30 minutes in lysis buffer (50 mM Tris, pH 8.8, 100 mM sodium chloride, 5 mM magnesium chloride, 0.5% nonylphenol ethoxylate (NP-40), 1 mM dithiothreitol (DTT), 250 IU / mL benzonuclide, 1 mM phenylmethylsulfonyl fluoride (PMSF), and 1× complete protease inhibitor cocktail). The lysate was centrifuged at 13,000 rpm at 4°C for 15 minutes. The NP-40-soluble supernatant was collected as the SN fraction. The NP-40-insoluble pellet (PE) was washed with phosphate-buffered saline (PBS), resuspended in precipitation buffer (20 mM Tris, pH 8.0, 15 mM magnesium chloride, 1 mM dithiothreitol (DTT), 250 IU / mL benzonuclide, 1 mM phenylmethylsulfonyl fluoride (PMSF), and 1× complete protease inhibitor cocktail), incubated on ice for 30 minutes, and then boiled in a buffer containing 2% sodium dodecyl sulfate (SDS). The SN and PE fractions were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).

[0122] (2) Western blotting was performed using antibodies targeting Tau and phosphorylated Tau, AT8 and Ser396.

[0123] 2. Tissue Immunofluorescence Staining

[0124] (1) Tissue dewaxing: Place the tissue in xylene I and II for 30 minutes each;

[0125] (2) Hydration of tissue sections: Place the slides in 100% I, 100% II, 95% I, 95% II, 75%, and 50% alcohol solutions for 5 minutes each;

[0126] (4) Wash with tap water once for 5 minutes; wash with distilled water three times for 5 minutes each; digest with 3% hydrogen peroxide solution for 8 minutes; wash with distilled water three times;

[0127] (5) Place in EDTA antigen retrieval solution, boil continuously on high heat in a microwave for 5 minutes, cool naturally to 80°C at room temperature, then boil repeatedly 3 times, cool naturally to 60°C at room temperature, and proceed to the subsequent steps;

[0128] (6) After cooling, wash with distilled water and 0.01M PBS solution three times, 5 minutes each time;

[0129] (7) Block with goat serum at room temperature for 1 hour;

[0130] (8) Blot dry the water stains, circle the edge of the tissue with a histochemical pen, add the primary antibody (diluted at 1:100), and incubate in a refrigerator at 4°C overnight (about 14 hours); place at room temperature the next day and allow to warm naturally for 10 minutes; rinse three times with 0.01M PBS solution;

[0131] (9) Add secondary antibody working solution (completely cover) and incubate at room temperature for 1 hour; rinse 3 times with 0.01M PBS solution; then stain the nucleus and seal the slide;

[0132] (10) Observe the staining using a fluorescence microscope.

[0133] 3. Learning and cognitive level test

[0134] (1) The Morris Water Maze test is a classic behavioral experiment for evaluating the spatial learning and memory ability of mice. The experimental preparation includes a water maze apparatus (a circular pool with a diameter of 1-1.5 meters and a height of 50-60 cm, filled with opaque water, and a water temperature of 22±1℃), a hidden platform (10-15 cm in diameter, 1-1.5 cm below the water surface), surrounding fixed visual markers and a video tracking system. The mice need to adapt to the laboratory environment for 3 days in advance to reduce stress. The experimental steps are divided into an adaptation phase (mice swim freely for 60 seconds to familiarize themselves with the environment, which is optional), a learning phase (4-7 consecutive days, 3 training sessions per day, starting from the other three quadrants where the platform is located and put into the water. The latency to find the hidden platform is recorded, with a maximum of 60 seconds. If it is not found, it is guided to the platform and stay for 15 seconds), a spatial exploration test (the platform is removed on the 9th day, and the mice swim freely for 60 seconds. The time spent in the target quadrant and the number of times the original platform is crossed are recorded) and a visual platform test (to verify the visual and motor abilities of the mice, which is optional). Precautions included maintaining consistent ambient lighting and sound levels, cleaning the pool to avoid odor disturbances, limiting swimming time to prevent stress on the mice, and drying and keeping the mice warm after the experiment. Mice were recorded and tracked using a video camera connected to automated tracking software (SMART 3.0, panlab Harvard apparatus). Data analysis focused on trends in latency reduction during the learning phase and time spent in the target quadrant during the exploration phase.

[0135] (2) Novel object recognition. First, the mice were allowed to acclimate to the environment in an empty open maze for 10 minutes. After 24 hours, the mice were placed in the center of the maze with two identical objects placed 5 cm away from the wall and allowed to explore these objects for 10 minutes. After 12 hours, the mice were placed in the center of the maze with the same two identical objects placed and allowed to explore for 5 minutes. After 3 hours, the mice were placed in the center of the maze with a familiar object and a novel object placed and allowed to explore for 5 minutes. The mice were recorded and tracked using a camera connected to automatic tracking software (SMART 3.0-panlab Harvard apparatus). The preference index was calculated by dividing the time spent exploring the novel object by the sum of the time spent exploring the novel object and the familiar object, and then multiplying the quotient by 100 to convert it into a percentage.

[0136] (3) Y-maze. Before the experiment, a Y-shaped maze apparatus consisting of three arms of equal length (usually 30-50 cm long, 5-15 cm wide, and 15-25 cm high) at a 120-degree angle must be prepared. Different visual or tactile marks can be placed at the end of each arm as spatial cues. The experimental environment must be kept quiet, with uniform lighting and no odor interference. The mouse was placed in the center of the three-arm Y-maze and tracked for 5 minutes. The score of spontaneous alternation behavior was calculated according to the following formula: Spontaneous alternation percentage = number of spontaneous alternations / (total number of arm entries-2) × 100%. Spontaneous alternation behavior is defined as the proportion of the number of entries into an arm different from the previous two choices to the total number of alternation opportunities.

[0137] Table 2 ILDR3 deficiency leads to aggravated Tau pathology

[0138]

[0139] Experimental results: Figure 7 As shown, ILDR3-deficient mice showed aggravated Tau pathology and decreased learning and cognitive abilities (Table 2, Figure 7 Tau levels in cerebrospinal fluid increased ( Figure 7 Middle C), Tau and phosphorylated Tau levels increased in hippocampal tissue ( Figure 7 Middle D), glial cell activation ( Figure 7 Middle E). Aβ levels are increased in the hippocampus of ILDR3-deficient mice ( Figure 7 Middle D).

[0140] These results indicate that ILDR3 deficiency can lead to aggravation of Tau-related diseases and abnormal accumulation of Aβ.

[0141] Experimental Example 7: ILDR3 Overexpression Delays the Progression of Tau Pathology

[0142] 1. Animal Experimental Protocol

[0143] PS19 mice were injected with AAV-ILDR3 (NM_017405.2) to overexpress the mouse ILDR3 gene or AAV-CTL control virus at 5 months of age. Tau pathology progression was assessed at 14 months of age. AAV-ILDR3 overexpression and AAV-CTL control viruses were packaged and prepared by Hanbio Biotech. Each mouse was injected with 3×10 11 vg.

[0144] 2. The detection of Tau pathology was consistent with the method described in Experimental Example 6.

[0145] Experimental results:

[0146] Table 3 ILDR3 overexpression delays Tau pathology progression

[0147]

[0148] like Figure 8 As shown, compared with the control virus AAV-CTL injected mice, the AAV-ILDR3 overexpression virus injected mice showed significantly reduced Tau pathology, that is, the learning and cognitive levels were higher than those of the control group (Table 3, Figure 8 Tau levels in cerebrospinal fluid decreased ( Figure 8 Middle D), Tau and phosphorylated Tau levels were reduced in hippocampal tissue ( Figure 8 Middle E, F), glial cell activation decreased ( Figure 8 Middle G).

[0149] These results indicate that ILDR3 and / or increasing ILDR3 expression can delay the progression of Tau-related diseases.

[0150] Experimental Example 8: ILDR3 Overexpression Improves Tau Pathology and Neurological Damage Caused by Tangled Tau

[0151] 1. At 4 months of age, PS19 mice were injected stereotaxically into the hippocampus (relative to the anterior bregma, horizontal distance -2.5 mm; anterior-posterior distance ±2.0 mm; vertical distance -1.8 mm), 2×10 10 vg AAV-ILDR3 (NM_017405.2) overexpressing the mouse ILDR3 gene or AAV-CTL control virus were co-injected with tangled Tau (PFF P301L Tau, 2 μg / μL, 2.5 μL), and the progression of Tau pathology was detected after 2 months.

[0152] 2. The detection method of Tau pathology is consistent with the method described in Experimental Example 6.

[0153] Experimental results:

[0154] Table 4 ILDR3 overexpression improves Tau pathology and neural damage caused by tau tangles

[0155]

[0156] like Figure 9 As shown, compared with the control virus AAV-CTL injected mice, the AAV-ILDR3 overexpression virus injected mice showed significantly reduced Tau pathology, that is, the learning and cognitive levels were higher than those of the control group (Table 4, Figure 9 Tau levels in cerebrospinal fluid decreased ( Figure 9 Middle D), Tau and phosphorylated Tau levels were reduced in hippocampal tissue ( Figure 9 (E, F).

[0157] This suggests that ILDR3 and / or increasing ILDR3 expression can treat and improve Tau-related diseases.

[0158] In summary, ILDR3 protein expression levels in brain tissues with tau-related diseases are significantly lower than in normal tissues; overexpression of ILDR3 promotes tau degradation and inhibits tau phosphorylation and aggregation; overexpression of ILDR3 enhances tau processing by microglia and astrocytes; and overexpression of ILDR3 can slow the progression of tau pathology and ameliorate the learning and cognitive decline caused by tau pathology. ILDR3 deficiency, on the other hand, leads to exacerbated tau pathology and decreased learning and cognitive abilities. Therefore, ILDR3 could be used as a drug, drug target, or gene therapy target for the prevention and / or treatment of tau-related diseases, providing new research foundations and intervention strategies for the prevention and treatment of tau-related diseases.

[0159] The foregoing description is merely a preferred experimental example of the present invention and is not intended to limit the present invention. Those skilled in the art will readily appreciate that various modifications and variations are possible. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention are intended to be within the scope of protection of the present invention.

Claims

1. Use of an expression promoter of ILDR3 gene and / or ILDR3 protein in the preparation of a drug for preventing and / or treating Alzheimer's disease, characterized in that: The expression promoter of the ILDR3 gene and / or ILDR3 protein includes any one of a recombinant expression vector containing the ILDR3 gene and an LNP liposome containing the ILDR3 gene.

2. The use according to claim 1, characterized in that The recombinant expression vector includes any one of a eukaryotic expression plasmid, adenovirus, adeno-associated virus, retrovirus, and homologous recombination vector.

3. The use according to claim 2, characterized in that The recombinant expression vector is a lentivirus.

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