Use of vascular-derived bace1 as a therapeutic target for diseases associated with small vessel injury in the brain

Abstract

The present application relates to the use of vascular BACE1 as a therapeutic target for diseases related to cerebral small vessel injury. Specifically, it relates to the use of a substance that reduces the expression level of the beta-secretase (BACE1) gene and / or an inhibitor of the enzymatic activity of BACE1 protein in the preparation of a drug for treating or alleviating cerebrovascular disease, wherein the disease related to cerebral small vessel injury is preferably Alzheimer's disease, amyloid cerebral small vessel disease, and vascular dementia. The inventors have found that an increase in the content of BACE1 can cleave the tight junction protein Occludin, resulting in the degradation and loss of tight junction proteins between vascular endothelial cells, causing the structure and function of the blood-brain barrier to be damaged, ultimately leading to cerebral microbleeds and cognitive dysfunction and other pathological phenotypes of diseases related to cerebral small vessel injury; inhibition of the enzymatic activity of BACE1 can improve these pathological phenotypes of diseases related to cerebral small vessel injury.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine, and particularly relates to the use of vascular BACE1 as a therapeutic target for cerebral small vessel disease. BACKGROUND

[0002] Cerebral vascular disease has become the first disease that seriously endangers human health with four characteristics of high incidence, high disability rate, high mortality and high recurrence rate. As an important part of cerebral vascular disease, cerebral small vessel disease has attracted the attention of scientific research and medical workers. Cerebral small vessel injury related disease is a general term for vascular diseases based on neuroanatomy, which refers to the clinical, cognitive, imaging and pathological manifestations of various lesions of cerebral arterioles, perforating arteries, capillaries and small venules. Cerebral small vessel injury related disease exists in many clinical diseases, such as Alzheimer's disease, amyloid cerebral small vessel disease, vascular dementia, etc.

[0003] The pathogenesis of cerebral small vessel injury related disease has not been clear so far. The core brain injury mechanism related to cerebral small vessel injury related disease is usually considered to be cerebral ischemia, or structural or functional small artery stenosis or occlusion. However, small artery occlusion may be a late result of cerebral small vessel injury related disease, and cannot explain early pathological changes. Some scholars believe that endothelial dysfunction and blood-brain barrier damage may be the early pathophysiological changes of cerebral small vessel injury related disease. Studies have shown that endothelial injury leads to increased vascular permeability, and intravascular components penetrate into the vascular wall and perivascular tissue, causing vascular wall injury and inflammatory response, demyelination, glial scar, vascular wall proliferation and hardening thickening, etc. Pathological changes, to the late stage, the lumen is narrowed and occluded, the cerebral small vessel disease arteriole tortuosity increases, arteriosclerosis, arteriopathy segmental dysplasia, blood vessel number or density decreases, and finally forms cerebral small vessel injury related disease.

[0004] The maintenance and operation of mammalian central nervous system function depends on the integrity of the structure and function of the blood brain barrier (BBB). As the first barrier for exogenous substances to enter the central nervous system, the blood brain barrier can effectively block toxic substances in the blood from entering the brain tissue, and plays an important role in the normal functioning of the central nervous system. As an important anatomical structure basis of the central nervous system, its formation and structural stability is the main guarantee for maintaining a good living environment for brain nerve cells. The structure of the blood brain barrier mainly includes: 1) brain microvessel endothelial cells (BMEC) and tight junctions (TJs) between cells without window pores, which constitute the first barrier of the blood brain barrier; 2) enzyme barrier (composed of extracellularly connected basement membrane of brain microvessel endothelial cells and nucleotide enzymes and some non-specific choline esterase substances), which constitutes the second barrier of the BBB; 3) the basement membrane is distributed with astrocytes, and its terminal foot constitutes the third barrier of the blood brain barrier.

[0005] Tight junctions (TJs) are the material basis for maintaining the integrity of the basic structure and function of the BBB. TJs are a protein complex composed of multiple proteins, including transmembrane proteins, cytoplasmic attachment proteins, connecting adhesion molecules and cytoskeleton proteins. Among them, the transmembrane proteins are Occludins and the Claudins family on endothelial cells; these transmembrane proteins are connected to the cytoskeleton through cytoplasmic attachment proteins ZO-1 / ZO-2 / ZO-3. These tight junction molecular elements interact with each other and polymerize with the tight junction proteins on the adjacent cell membrane, promoting the fusion of the cell membrane locally and forming the complex three-dimensional network ultrastructure of tight junctions under electron microscopy. Occludin is the main component of intercellular TJs, and endothelial cells close the intercellular space through Occludin, and together with ZO-1 and other tight junction-related proteins, they form the basic structure of TJs. The disruption of tight junctions is related to the dephosphorylation of serine and threonine residues of Occludin, and the absence of Occludin leads to the destruction of the cytoskeleton and the reduction of cell processes. These phenomena suggest that Occludin plays a crucial role in maintaining the integrity of the structure and function of the blood brain barrier.

[0006] BACE1 (β-site APP cleaving enzyme, β-secretase) belongs to the aspartic protease family. BACE1 mainly exists in nerve cells. Like typical aspartic proteases such as pepsin, BACE1 has a large number of β-sheet structures and catalyzes the peptide chain of the substrate with two key aspartic acid catalytic residues close to each other. It is found that the substrates of BACE1 include amyloid precursor protein (APP), neuregulin-1 (NRG1), etc. The substrates of BACE1 are involved in many pathological processes and are important disease treatment targets. For example, in the pathological process of Alzheimer's disease (AD), neuroinflammatory plaques are mainly formed by β-amyloid protein (Aβ), which is produced by continuous cleavage of APP by BACE1 and γ-secretase (γ-secretase). BACE1 is considered to be the rate-limiting enzyme in this process, and therefore BACE1 inhibitors are considered to be useful for treating AD.

[0007] BACE1 is associated with various brain diseases. In addition to AD, studies have shown that the expression level of BACE1 in the brain of patients with a type of cerebrovascular disease, cerebral amyloid angiopathy (CAA), increases, and the expression level of endothelial cell tight junction protein decreases, accompanied by pathological phenotypes related to blood-brain barrier damage such as cerebral hemorrhage. Further studies have shown that vascular cells also express BACE1, and factors such as inflammation and oxidative stress, which are risk factors for blood vessels, can increase the expression of BACE1. Therefore, the above evidence suggests that BACE1 can be a downstream effect target of vascular risk factors, acting on the endothelial cells of the brain blood vessels, destroying the tight junction structure of the vascular endothelial cells, thereby affecting the function of the blood-brain barrier and ultimately affecting the progression of brain small vessel injury-related diseases.

[0008] So far, there is no research result to confirm that BACE1 can be used as a therapeutic target for brain small vessel injury-related diseases, and it is of great significance to find a drug that selectively regulates BACE1 gene or protein for the treatment of brain small vessel injury-related diseases. SUMMARY

[0009] The present application is to solve the problems in the prior art. The present inventors have found that vascular BACE1 can specifically cleave tight junction protein Occludin. The increase of BACE1 expression level can cleave tight junction protein Occludin, thereby causing damage and loss of tight junction of endothelial cells themselves and between endothelial cells, causing vascular damage and blood brain barrier dysfunction, and ultimately leading to brain hemorrhage and cognitive dysfunction and other brain small vessel damage related disease pathological phenotypes. Meanwhile, the inhibition of BACE1 enzyme activity can improve these brain small vessel damage related disease pathological phenotypes. The decrease of BACE1 expression level can alleviate the damage of tight junction protein Occludin structure, thereby alleviating vascular damage and blood brain barrier dysfunction. Therefore, BACE1 is a potential new target for treating or alleviating cerebrovascular diseases. The present inventors have completed the present application on this basis.

[0010] To this end, the present application provides the use of a substance for reducing the expression level of a beta-secretase (BACE1) gene and / or an inhibitor of BACE1 protein enzyme activity in the preparation of a medicament for treating or alleviating cerebrovascular diseases.

[0011] In some embodiments of the present application, the brain small vessel damage related diseases mainly include Alzheimer's disease, amyloid cerebral small vessel disease and vascular dementia.

[0012] In some embodiments of the present application, the medicament comprises an active ingredient, which is a substance for reducing the expression level of a BACE1 gene and / or an inhibitor of BACE1 protein enzyme activity.

[0013] In some embodiments of the present application, the substance for reducing the expression level of a BACE1 gene comprises a specific transcription inhibitor of a BACE1 gene.

[0014] In some embodiments of the present application, the specific transcription inhibitor of a BACE1 gene refers to an inhibitor that specifically inhibits the transcription process of a BACE1 gene. By inhibiting the transcription of a BACE1 gene, the expression level of a BACE1 gene and the level of a BACE1 protein in a patient are reduced, the cleavage of tight junction protein Occludin is alleviated, and for example, an RNAi fragment or an siRNA fragment targeting a BACE1 gene, or a CRISPR gRNA promoting the degradation of a BACE1 messenger RNA.

[0015] In some embodiments of the present application, the inhibitor of a BACE1 protein can be C3 (CAS 797035-11-1) or MK-8931 (CAS 1286770-55-5) and other inhibitors of a BACE1 protein known to those skilled in the art.

[0016] The second aspect of the present application provides a drug for treating or alleviating a disease related to cerebral small vessel damage, wherein the drug comprises an active ingredient, the active ingredient being a substance that reduces the expression level of a BACE1 gene and / or an inhibitor of the enzymatic activity of a BACE1 protein.

[0017] In some embodiments of the present application, the substance that reduces the expression level of a BACE1 gene comprises a specific transcription inhibitor of a BACE1 gene, such as an RNAi fragment or an siRNA fragment targeting a BACE1 gene, or a CRISPR gRNA that promotes the degradation of a BACE1 messenger RNA.

[0018] In some embodiments of the present application, the drug further comprises a pharmaceutically acceptable carrier, which is generally non-toxic to a recipient at the doses and concentrations employed, including but not limited to: buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (e.g., octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as poly(vinylpyrrolidone); amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions such as sodium; metal complexes (e.g., zinc proteins complexes); and / or non-ionic surfactants such as polyethylene glycol (PEG).

[0019] The third aspect of the present application provides an agent or a kit for treating or alleviating a disease related to cerebral small vessel damage, wherein the agent or the kit comprises a substance that reduces the expression level of a BACE1 gene and / or an inhibitor of the enzymatic activity of a BACE1 protein.

[0020] In some embodiments of the present application, the substance that reduces the expression level of a BACE1 gene comprises a specific transcription inhibitor of a BACE1 gene, such as an RNAi fragment and / or an siRNA fragment targeting a BACE1 gene.

[0021] In some embodiments of the present application, the inhibitor of the enzymatic activity of a BACE1 protein is C3 (CAS 797035-11-1) or MK-8931 (CAS 1286770-55-5).

[0022] The inventors found that the increase of BACE1 protein expression level can specifically cleave Occludin, resulting in the damage and loss of tight junction structure between vascular endothelial cells. The present application further provides an animal model with vascular endothelial cell-specific overexpression of BACE1 protein. Further, the animal model exhibits pathological phenotypes related to cerebral small vessel injury, such as damage and loss of tight junction structure in brain vascular endothelial cells, blood brain barrier damage, cerebral microhemorrhage, and learning behavior and cognitive dysfunction. BACE1 can specifically cleave Occludin, thereby destroying the tight junction structure, causing vascular injury and vascular barrier damage, and ultimately forming pathological phenotypes related to cerebral small vessel injury-related diseases. Reducing the expression level of BACE1 or inhibiting the enzyme activity of BACE1 can alleviate vascular injury. Therefore, BACE1 can be used as a therapeutic target for treating cerebral small vessel injury-related diseases. BRIEF DESCRIPTION OF DRAWINGS

[0023] The present application will be described in detail below with reference to the accompanying drawings

[0024] Figure 1 : Western blot result figure and semi-quantitative analysis figure of BACE1-specific cleavage of Occludin to generate N-terminal and C-terminal fragments (7 repeated experiments, and 1 experiment is shown in the left figure);

[0025] Figure 2 : Western blot result figure and semi-quantitative analysis figure of Occludin cleaved by HA-BACE1 of different concentrations (7 repeated experiments, and 1 experiment is shown in the left figure);

[0026] Figure 3 : Western blot result figure and semi-quantitative analysis figure of inhibition of Occludin cleaved by HA-BACE1 by BACE1 inhibitor C3 of different concentrations (7 repeated experiments, and 1 experiment is shown in the left figure);

[0027] Figure 4 : Western blot result figure and semi-quantitative analysis figure of inhibition of Occludin cleaved by BACE1 by si-BACE1 (7 repeated experiments, and 3 repeated experiments are shown in the left figure);

[0028] Figure 5 : Western blot result figure and semi-quantitative analysis figure of cleavage of Occludin in CRISPR-Cas9 BACE1 KO (knockout) cells (12 repeated experiments, and 2 repeated experiments are shown in the left figure);

[0029] Figure 6: Western blot results of detecting Occludin expression levels in the brains of BACE1 KO mice and its semi-quantitative analysis (8 replicate experiments, the left figure shows 3 of the replicate experiments);

[0030] Figure 7 Figure 1 shows the Western blot results of BACE1 cleaving different mutant sequences of Occludin (3 replicate experiments). Figure A shows the Western blot results of BACE1 cleaving four single-point mutations of Occludin (L87G, A88G, W89G, D90G). Figures BC show the Western blot results of BACE1 cleaving two double-point mutations of Occludin (L87G / A88G, W89G / D90G). Figure D shows the Western blot results of BACE1 cleaving deletion mutation of Occludin (87-90 deletion mutation).

[0031] Figure 8 Western blot results showing BACE1 cleavage of endogenous Occludin in HUVEC cells and its effect on other endogenous tight junction proteins;

[0032] Figure 9 The effect of different BACE1 concentrations on in vitro endothelial cell permeability (6 replicate experiments);

[0033] Figure 10 : Western blot results showing the effect of si-occludin on the action of BACE1 on other tight junction proteins;

[0034] Figure 11 : Western blot results showing the effect of lysosomal inhibitor A1 on the effect of BACE1 on other tight junction proteins;

[0035] Figure 12 Colocalization results of tight junction proteins (Claudin-1, ZO-1) with lysosome EEA1 and autophagosome LC3 (points indicated by arrows);

[0036] Figure 13 Western blot results of BACE1 expression levels in various tissues of WT mice and VE-BACE1 mice;

[0037] Figure 14 Figure 1: Results of co-localization staining of vascular endothelial cells with BACE1 in WT mice and VE-BACE1 mice.

[0038] Figure 15 Western blot results of tight junction protein expression levels in the brains of WT mice and VE-BACE1 mice (3 replicates);

[0039] Figure 16 Immunofluorescence images used to detect the expression levels of vascular tight junction protein in the brains of WT mice and VE-BACE1 mice;

[0040] Figure 17 Immunofluorescence images of immune endothelial cells in the brains of WT mice and VE-BACE1 mice (endothelial cell marker CD31);

[0041] Figure 18 Immunofluorescence images of endothelial damage in the brains of WT mice and VE-BACE1 mice (endothelial damage marker caveolin-1);

[0042] Figure 19 Immunofluorescence images of the terminal feet of astrocytes in the brains of WT mice and VE-BACE1 mice (AQP4, a marker for the terminal feet of astrocytes);

[0043] Figure 20 Immunofluorescence images of the brains of WT mice and VE-BACE1 mice (peripheral cell marker Desmin);

[0044] Figure 21 Immunofluorescence images of all blood vessels in the brains of WT mice and VE-BACE1 mice (based on the basement membrane marker Laminin);

[0045] Figure 22 Immunofluorescence images of all blood vessels in the brains of WT mice and VE-BACE1 mice (pericytic cell marker PDGFRβ);

[0046] Figure 23 Immunofluorescence images of vascular smooth muscle cells in the brains of WT mice and VE-BACE1 mice (vascular smooth muscle cell marker SMA);

[0047] Figure 24 Immunofluorescence images of small arteries and venules in the brain of WT mice and VE-BACE1 mice (vWF markers for arteries and venules);

[0048] Figure 25 Immunofluorescence images of small veins and capillaries in the brains of WT mice and VE-BACE1 mice (molecular marker MCT1 for small veins and capillaries);

[0049] Figure 26 Immunofluorescence images of cerebral veins (marker VCAM-1) in WT mice and VE-BACE1 mice;

[0050] Figure 27Western blot results and semi-quantitative analysis of fibrin(ogen) content in the brains of WT mice and VE-BACE1 mice (7-12 replicate experiments, the left figure shows 3 of the replicate experiments);

[0051] Figure 28 Immunohistochemical staining images of fibrin(ogen) in the brains of WT mice and VE-BACE1 mice;

[0052] Figure 29 Immunohistochemical staining images of the brains of WT mice and VE-BACE1 mice (GFAP-labeled astrocytes)

[0053] Figure 30 Immunohistochemical staining images of microglia in the brains of WT mice and VE-BACE1 mice (iba-1 labeled microglia);

[0054] Figure 31 Immunohistochemical staining images of neurons in the brains of WT mice and VE-BACE1 mice (Nuen-labeled neurons);

[0055] Figure 32 Immunohistochemical staining images of the presynaptic brains of WT mice and VE-BACE1 mice (SNAP25 presynaptic markers);

[0056] Figure 33 Immunohistochemical staining images of postsynaptic markers in the brains of WT mice and VE-BACE1 mice (PSD-95 postsynaptic markers);

[0057] Figure 34 : Results of the experiment to detect the blood-brain barrier permeability of VE-BACE1 mice (6 repeated experiments);

[0058] Figure 35 Quantitative analysis of Prussian blue staining results in the brains of VE-BACE1 mice (4 replicate experiments);

[0059] Figure 36 Results of the Barnes maze experiment for VE-BACE1 mice (11 age-matched mice in each group, showing the time to find the hole during training (top), day 5 (middle), and day 12 (bottom)).

[0060] Figure 37 Western blot analysis of changes in tight junction protein levels in brain blood vessels in WT mice and VE-BACE1 mice after three months of administration of 1.5 mg / kg BACE1 enzyme activity inhibitor MK-8931 or DMSO (the figure shows three replicates of the experiment).

[0061] Figure 38Western blot semi-quantitative analysis of Occludin FL content in the brains of WT mice and VE-BACE1 mice after three months of administration of 1.5 mg / kg BACE1 enzyme activity inhibitor MK-8931 or DMSO (6 replicate experiments).

[0062] Figure 39 Western blot semi-quantitative analysis of Occludin CTF content in the brains of WT mice and VE-BACE1 mice after three months of administration of 1.5 mg / kg BACE1 enzyme activity inhibitor MK-8931 or DMSO (6 replicate experiments).

[0063] Figure 40 Semi-quantitative Western blot analysis of the content of tight junction protein ZO-1 in the brains of WT mice and VE-BACE1 mice after three months of administration of 1.5 mg / kg BACE1 enzyme activity inhibitor MK-8931 or DMSO (6 replicate experiments).

[0064] Figure 41 Semi-quantitative Western blot analysis of JAM-A content in the brains of WT mice and VE-BACE1 mice after three months of administration of 1.5 mg / kg BACE1 enzyme activity inhibitor MK-8931 or DMSO (6 replicate experiments).

[0065] Figure 42 Western blot semi-quantitative analysis of Clauin-1 content in the brains of WT mice and VE-BACE1 mice after three months of administration of 1.5 mg / kg BACE1 enzyme activity inhibitor MK-8931 or DMSO (6 replicate experiments).

[0066] Figure 43 : The total movement distance of WT mice and VE-BACE1 mice in open field experiments after administration of 1.5 mg / kg BACE1 enzyme activity inhibitor MK-8931 or DMSO (10 age-matched mice in each group);

[0067] Figure 44 : Results of open field test reflecting anxiety in WT mice and VE-BACE1 mice after administration of 1.5 mg / kg BACE1 enzyme activity inhibitor MK-8931 or DMSO (10 age-matched mice in each group);

[0068] Figure 45The results of the total number of arm entries in the Spontaneous Y-maze test in WT mice and VE-BACE1 mice after administration of 1.5 mg / kg BACE1 enzyme activity inhibitor MK-8931 or DMSO (left) and the percentage of correct entry order (right) (10 age-matched mice per group).

[0069] Figure 46 Figure 1: Results of a novel object recognition experiment in WT mice and VE-BACE1 mice after administration of 1.5 mg / kg body weight of the BACE1 enzyme activity inhibitor MK-8931 or DMSO (10 age-matched mice in each group).

[0070] Figure 47 : WT mice and VE-BACE1 mice administered 1.5 mg / kg body weight of BACE1 enzyme activity inhibitor MK-8931 or DMSO on day 5 of the Barnes maze test reflecting short-term memory (10 age-matched mice in each group).

[0071] Figure 48 WT mice and VE-BACE1 mice administered 1.5 mg / kg body weight of BACE1 enzyme activity inhibitor MK-8931 or DMSO on day 12 of the Barnes maze test, reflecting the results of long-term memory (10 age-matched mice in each group). Detailed Implementation

[0072] The technical solution of the present invention is illustrated below through specific embodiments. However, these embodiments are for illustrative purposes only and do not imply that the scope of the present invention is limited thereto.

[0073] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0074] Unless otherwise specified, all experimental materials and reagents used in the following examples are commercially available.

[0075] Example 1: BACE1-specific cleavage of Occludin.

[0076] 1. Experimental procedure for BACE1-specific cleavage of Occludin

[0077] To explore the relationship between human BACE1 and human Occludin, HEK293 cells stably transfected with both HA-BACE1 and Flag-Occludin-Myc plasmids were constructed. Western blot analysis was used to detect changes in the full-length, N-terminus, and C-terminus of Occludin (see [link to article]). Figure 1Meanwhile, by transfecting HEK293 cells stably transfected with both HA-BACE1 and Flag-Occludin-Myc plasmids with different concentrations of HA-BACE1 plasmid and adding different concentrations of the BACE1 inhibitor C3, the changes in the N-terminus and C-terminus of Occludin were further detected by Western blot (see [link to article]). Figure 2 and 3 On the other hand, stable BACE1 knockdown and knockout cell lines were constructed using siRNA and CRISPR-Cas9 technologies, respectively. These cell lines were then transfected with the Flag-Occludin-Myc plasmid, and Western blot analysis was used to detect changes in the N-terminus and C-terminus of Occludin (see [link to relevant documentation]). Figure 4 and 5 Furthermore, stable BACE1 knockout mice (purchased from Jackson Laboratories) were constructed, and the expression of Occludin in the mouse brain was detected using Western blot technology.

[0078] 2. Experimental Materials

[0079] BACE1 knockout mice were purchased from Jackson Laboratories; HEK293 cells and cell culture reagents were purchased from GIBICO (Carlsbad, CA); BACE1 inhibitor C3 (Merck); Occludin cDNAs were purchased from Addgene; siRNA was purchased from GenePharma.

[0080] The antibodies are as follows:

[0081] Primary resistance:

[0082] anti-Myc tag antibody (Santa Cruz, SC-40 / 9E10);

[0083] anti-Flag tag antibody (Sigma, F1804);

[0084] Anti-Occludin antibodies (anti-C-terminal Occludin (269-522aa) antibody (Proteintech, 13409-1-AP); anti-N-terminal Occludin (1-100aa) antibody (Abcam, ab167161));

[0085] anti-GAPDH antibody (MAB374, Millipore);

[0086] Secondary antibody:

[0087] HRP-anti-rabbit IgG (Jackson Research);

[0088] HRP-anti-mouse IgG (Jackson Researc).

[0089] Carrier:

[0090] HA-PKH3 (Addgene)

[0091] pCMV26(Addgene)

[0092] 3. Specific operations

[0093] In this study, all cells were cultured in continuous monolayers in DMEM medium containing 10% FBS, 2 mM L-glutamate, 100 μg / ml penicillin, and 100 μg / ml streptomycin at 37°C and 5% CO2. HA-BACE1 and Flag-Occludin-Myc were transfected into HEK293 cells using Lipofectamine 2000. siRNA transfection was performed using LipofectamineRNAiMAX (Life Technologies).

[0094] (1) The HA-BACE1 plasmid (where BACE1 is the full-length sequence) was obtained by PCR amplification using human BACE1 cDNA (NM_012104.6) and vector HA-PKH3; the Flag-Occludin-Myc plasmid (where Occludin is the full-length sequence) was obtained by PCR amplification using human Occludin cDNA (NM_001205254.2) and vector pCMV26.

[0095] (2) Using CRISPR-Cas9 technology, targeting the human BACE1 gene, the BACE1 knockout HEK293 cell line was obtained.

[0096] CRISPR-Cas9 BACE1 knockout sgRNA sequence (three sequences used in combination):

[0097] sgBACE1-1: 5'-GGATCCGGAGCCCGCTACAT-3' (SEQ ID NO: 1);

[0098] sgBACE1-2: 5'-CGGGCTCTTCGTCGGTCTCC-3' (SEQ ID NO: 2);

[0099] sgBACE1-3: 5'-TACTACGTGGAGATGACCGT-3' (SEQ ID NO: 3).

[0100] (3) HEK293 cells co-transfected with HA-vector (i.e., HA-PKH3, empty vector, abbreviated as HA in the figure) or HA-BACE1 and Flag-Occludin-Myc: The experiment was divided into control group (co-transfected with HA-vector and Flag-Occludin-Myc) and experimental group (co-transfected with HA-BACE1 and Flag-Occludin-Myc).

[0101] (4) Two 6-well HEK293 cells were first transfected with 2 μg Flag-Occludin-Myc. After 24 hours of transfection, they were combined and passaged to form six 6-well HEK293 cells. Then, they were transfected with HA or different concentrations of HA-BACE1. The experiment was divided into six groups:

[0102] The control group (co-transfected with 2 μg Flag-Occludin-Myc and 2.5 μg HA-vector (i.e., HA-PKH3, empty vector, abbreviated as HA in the figure))

[0103] Experimental group 1 (co-transfected with 2 μg Flag-Occludin-Myc and 0.5 μg HA-BACE1),

[0104] Experimental group 2 (co-transfected with 2 μg Flag-Occludin-Myc and 1.0 μg HA-BACE1),

[0105] Experimental group 3 (co-transfected with 2 μg Flag-Occludin-Myc and 1.5 μg HA-BACE1),

[0106] Experimental group 4 (co-transfected with 2 μg Flag-Occludin-Myc and 2.0 μg HA-BACE1),

[0107] Experimental group 5 (co-transfected with 2 μg Flag-Occludin-Myc and 2.5 μg HA-BACE1).

[0108] (5) Experiment to detect the effect of different concentrations of BACE1 inhibitor C3 on BACE1 cleavage of Occludin: HEK293 cells (cells that express BACE1 protein) were transfected with Flag-Occludin-Myc and different concentrations of BACE1 inhibitor C3 were added. The experiment was divided into five groups: control group (transfected with Flag-Occludin-Myc and treated with 15 μM DMSO (as a blank control of C3) for 4 hours), experimental group 1 (transfected with Flag-Occludin-Myc and treated with 5 μM C3 for 4 hours), experimental group 2 (transfected with Flag-Occludin-Myc and treated with 10 μM C3 for 4 hours), and experimental group 3 (transfected with Flag-Occludin-Myc and treated with 15 μM C3 for 4 hours).

[0109] (6) Experiment to detect the effect of si-BACE1 on BACE1 cleavage of Occludin: HEK293 cells were transfected with Flag-Occludin-Myc and siRNA was added. The experiment was divided into two groups: control group (transfected with Flag-Occludin-Myc and si-control was added) and experimental group (transfected with Flag-Occludin-Myc and si-BACE1 was added).

[0110] The si-BACE1 sequence is:

[0111] si-BACE1: 5'-CCCAAGACGACTGTTACAATT-3' (SEQ ID NO: 4);

[0112] si-control sequence: 5'-UUCUCCGAACGUGUCACGUTT-3'SEQ ID NO: 5 (obtained by randomly scrambling the oligonucleotide sequence of the above siRNA-BACE1).

[0113] (7) CRISPR-Cas9 BACE1 knockout HEK293 cell transfection with Flag-Occludin-Myc experiment: The experiment was divided into two groups: control group (WT HEK293 cells transfected with Flag-Occludin-Myc) and experimental group (CRISPR-Cas9 BACE1 knockout HEK293 cells transfected with Flag-Occludin-Myc).

[0114] (8) Experiment to detect the expression of Occludin in the brain of BACE1 KO mice: After anesthetizing BACE KO mice with sodium pentobarbital, they were perfused with 1×PBS, and the brains were completely removed with surgical instruments and stored at -80℃.

[0115] Cell samples were lysed and collected using cell lysis buffer (1% Nonidet P-40, 0.5% sodium deoxycholate, 1M Tris-HCl, 150mM NaCl, pH 7.5, and protease inhibitor Cocktail (Roche)). Cells were lysed at room temperature, and then an equal volume of 2×SDS electrophoresis loading buffer was added. The samples were then boiled in boiling water for 10 minutes to prepare SDS electrophoresis samples.

[0116] An appropriate amount of brain tissue from BACE1 KO mice was taken and homogenized in cell lysis buffer (phosphate buffer, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, and protease inhibitor cocktail) on ice using a Durns tissue homogenizer, sonicated for 4 minutes, and centrifuged at 160,000g for 30 minutes at 4°C.

[0117] The changes in Occludin-FL (full-length Occludin), Occludin-CTF (C-terminal fragment of Occludin), and Occludin-NTF (N-terminal fragment of Occludin) were detected using Western blot technology.

[0118] Western blotting procedure:

[0119] SDS-PAGE electrophoresis: Prepare an appropriate concentration of SDS electrophoresis gel according to the size of the protein to be tested. After loading equal amounts of the sample to be tested, perform electrophoretic separation (upper stacking gel constant voltage 90 volts, lower separating gel constant voltage 120 volts).

[0120] Transfer: After SDS-PAGE electrophoresis, transfer is performed (PVDF membrane is activated with methanol before use). The transfer time is different depending on the protein size at a constant current of 240 amperes (90-180 minutes). The transfer process is carried out in an ice-water bath.

[0121] Blocking: After the transfer is complete, place the protein-loaded membrane in 5% skim milk prepared with TBST buffer and block on a shaker at room temperature for 1 hour.

[0122] Primary antibody incubation: Prepare an appropriate concentration of primary antibody according to the instructions for use of the protein antibody or the results of preliminary experiments. After blocking, incubate the transfer membrane at room temperature for 3 hours and then at 4°C overnight.

[0123] Wash primary antibody: Wash 3 times with TBST buffer at room temperature on a shaker for 5 minutes each time;

[0124] Secondary antibody incubation: Incubate the membrane with the secondary antibody corresponding to the primary antibody on a shaker at room temperature for 40 minutes to 1 hour;

[0125] Washing secondary antibody: Wash with TBST buffer three times with shaking on a shaker at room temperature for 5 minutes each time;

[0126] Colorimetric assay: The protein content signal on the membrane was detected by colorimetric assay using an ECL imaging kit.

[0127] (8) Semi-quantitative analysis: ImageJ software was used to analyze the content of each target protein and its internal reference. The content of each target protein was divided by its internal reference content. The resulting value is the relative content of the target protein in each sample after internal reference correction. This value was then used to compare and analyze the samples to obtain the actual change results of the target protein content in different samples.

[0128] 4. Results Analysis

[0129] Depend on Figure 1 The results show that Occludin is a substrate for BACE1 cleavage, and BACE1 specifically cleaves Occludin to produce N-terminal and C-terminal fragments.

[0130] Depend on Figure 2 The results showed that with the increase of HA-BACE1 transfection concentration, the contents of Occludin NTF and CTF increased significantly, while the contents of Occludin FL decreased significantly, indicating that the increase of HA-BACE1 transfection concentration can enhance its cleavage effect on Occludin.

[0131] Depend on Figure 3 The results showed that the higher the concentration of BACE1 inhibitor C3, the less ocludin was cleaved, indicating that different concentrations of BACE1 inhibitor C3 could inhibit HA-BACE1 cleavage of ocludin, and that the cleavage effect of BACE1 inhibitor C3 on ocludin weakened with increasing concentration.

[0132] Depend on Figure 4 The results showed that the siRNA method reduced the protein expression level of BACE1, and the cleavage of Occludin by BACE1 was inhibited, indicating that the reduction of BACE1 protein expression level can inhibit its cleavage of Occludin.

[0133] Depend on Figure 5 The results showed that the cleavage of Occludin by BACE1 was inhibited in CRISPR-Cas9 BACE1 KO cells.

[0134] Depend on Figure 6 The results showed that the protein level of Occludin was significantly upregulated in the brains of BACE1 KO mice, while the levels of other tight junction proteins ZO-1, JAMA and Claudin-1 were not significantly changed.

[0135] Example 2: Sites where BACE1 cleaves Occludin protein.

[0136] 1. Experimental Procedure

[0137] Construct a 30-amino acid peptide fragment of human Occludin linked to biotin-

[0138] biotin-Occludin 81-110 :

[0139] biotin-ACVASTLAWDRGYGTSLLGGSVGYPYGGSG(Occludin 81-110 (SEQ ID NO: 6), the polypeptide fragment was co-incubated with BACE1 in vitro and then analyzed using LC-MS / LM technology. Mass spectrometry results without BACE1 treatment detected two fragments: biotin-Occludin. 81-110 and Occludin 90-110 LC-MS / LM results after BACE1 treatment showed biotin-Occludin 81-110 Peak value decreases while Occludin 90-110 The increase was 4 times, and three additional biotin-Occludin fragments were added. 81-87 biotin-Occludin 81-88 Occludin 88-110 and Occludin 89-110 Therefore, it is speculated that BACE1 cleaves Occludin to the L site. 87 / A 88 A 88 / W 89 and W 89 / D 90 Furthermore, plasmids with Occludin L87G, A88G, W89G, and D90G mutations (all mutated to G), as well as plasmids with L87G / A88G and W89G / D90G double-site mutations (all mutated to G), and Flag-Occludin-Myc were constructed. 87-90 The mutation sequence was deleted, and changes in Occludin-FL, Occludin-CTF, and Occludin-NTF were detected using cell transfection and Western blot techniques.

[0140] 2. Experimental Materials

[0141] HEK293 cells were cultured using GIBICO (Carlsbad, CA) reagents, and the cell culture conditions were the same as above; biotin-Occludin 81-110 The peptides were purchased from Genescript.

[0142] Peptide amino acid sequence: bio-ACVASTLAWDRGYGTSLLGGSVGYPYGGSG (SEQ ID NO: 6).

[0143] Primary antibodies: anti-Myc tag antibody (Santa Cruz, SC-40 / 9E10), anti-Flag tag antibody (Sigma, F1804), anti-Occludin antibody (anti-C-terminal Occludin (269-522aa) antibody (Proteintech, 13409-1-AP); anti-N-terminal Occludin (1-100aa) antibody (Abcam, ab167161)), anti-GAPDH antibody (MAB374, Millipore);

[0144] Secondary antibodies: HRP-anti-rabbit IgG (Jackson Research), HRP-anti-mouse IgG (Jackson Research).

[0145] Primers for constructing Flag-Occludin-Myc mutant sequences:

[0146] Regarding Flag-Occludin-Myc L87G:

[0147] LP: 5′-GGTGCCTGGGACAGAGGCTAT-3′ (SEQ ID NO: 7);

[0148] RP: 5′-CGTGGAGGCCACACAGGCAAA-3′ (SEQ ID NO: 8);

[0149] For Flag-Occludin-Myc A88G

[0150] LP: 5′-GGGGACAGAGGCTATGGAACTTCC-3′ (SEQ ID NO: 9);

[0151] RP: 5′-GGCAAGCGTGGAGGCC-3′ (SEQ ID NO: 10);

[0152] For Flag-Occludin-Myc W89G

[0153] LP: 5′-GGGGACAGAGGCTATGGAACTTCC-3′ (SEQ ID NO: 11);

[0154] RP: 5′-GGCAAGCGTGGAGGCC-3′ (SEQ ID NO: 12);

[0155] For Flag-Occludin-Myc D90G

[0156] LP: 5′-GGCAGAGGCTATGGAACTTCCCT-3′ SEQ ID NO: 13);

[0157] RP: 5′-CCAGGCAAGCGTGGAGGCC-3′ SEQ ID NO: 14);

[0158] For Flag-Occludin-Myc L87G / A88G

[0159] LP: 5′-GGCCTCCACGGGTGGCTGGGACAGAG-3′ SEQ ID NO: 15);

[0160] RP: 5′-ACACAGGCAAAGATGGCAATG-3′ SEQ ID NO: 16);

[0161] For Flag-Occludin-Myc W89G / D90G

[0162] LP: 5′-CACGCTTGCCGGGGGCAGAGGCTATG-3′ SEQ ID NO: 17);

[0163] RP: 5′-GAGGCCACACAGGCAAAGATG-3′ SEQ ID NO: 18);

[0164] For Flag-Occludin-Myc 87-90 deletion

[0165] LP: 5′-AGAGGCTATGGAACTTCCCTTTTA-3′ SEQ ID NO: 19);

[0166] RP: 5'-CGTGGAGGCCACACAGGCAAA-3' SEQ ID NO: 20).

[0167] 3. Specific operations

[0168] (1) LC-MS / LM technique to determine the BACE1 cleavage site of Occludin: 20 μg biotin-Occludin 81-110 The peptide was reacted with purified BACE1 in 50 mM sodium acetate (50% acetonitrile, 0.1% trifluoroacetic acid) at 37 °C and pH 4.5 for 12 h. The resulting system was separated by reversed-phase liquid chromatography-mass spectrometry (RPLC-MS) for 50 min at a flow rate of 8 μL / min. The mixture was separated using a linear gradient solution: 88% solvent A (ultrapure water containing 5% acetonitrile, 0.1% formic acid, and 0.01% trifluoroacetic acid) and 12% solvent B (85% acetonitrile, 10% isopropanol, 0.075% formic acid, and 0.0075% trifluoroacetic acid), and 80% solvent B.

[0169] (2) Using PCR technology, four single-site mutation Flag-Occludin-Myc (L87G, A88G, W89G, D90G) sequences, two double-site mutation Flag-Occludin-Myc (L87G / A88G, W89G / D90G) sequences, and Flag-Occludin-Myc were amplified from Occludin cDNA and different primers. 87-90 Deletion of the mutant sequence. The Flag-Occludin-Myc mutant sequence was transfected into HEK293 cells using Lipofectamine 2000.

[0170] (3) Detection of BACE1 splicing mutation Occludin expressed in HEK293 cells: The Flag-Occludin-Myc sequence or the obtained mutated Flag-Occludin-Myc sequence was stably transfected into HEK293 cells. The experiment was divided into: control group (transfected with WT Flag-Occludin-Myc sequence), experimental group: transfected with Flag-Occludin-Myc single-point mutation sequence (transfected with four single-point mutation sequences: L87G, A88G, W89G, D90G respectively), transfected with Flag-Occludin-Myc double-site mutation sequence (transfected with two double-site mutation sequences: L87G / A88G, W89G / D90G respectively), and transfected with Flag-Occludin-Myc... 87-90 Missing mutant sequence.

[0171] Cell samples were lysed and collected using cell lysis buffer (1% Nonidet P-40, 0.5% sodium deoxycholate, 1M Tris-HCl, 150mM NaCl, pH 7.5, protease inhibitor cocktail); cells were lysed at room temperature, and then an equal volume of 2×SDS electrophoresis loading buffer was added. The samples were then boiled in boiling water for 10 minutes to prepare SDS electrophoresis samples.

[0172] Changes in Occludin-FL, Occludin-CTF, and Occludin-NTF were detected using Western blot: the procedure for Western blotting was the same as in Example 1.

[0173] 4. Results Analysis

[0174] Figure 7 The results show that Flag-Occludin-Myc 87-90 The deletion significantly inhibited BACE1's cleavage of Occludin, indicating that the cleavage site does indeed exist in the amino acid region between positions 87 and 90. However, the single-site mutation at Flag-Occludin-Myc could not completely inhibit BACE1's cleavage of Occludin, while the double-site mutation at Flag-Occludin-Myc significantly inhibited BACE1's cleavage of Occludin. Therefore, it is confirmed that the site of BACE1's cleavage of Occludin is L... 87 / A 88 A 88 / W 89 and W 89 / D 90 .

[0175] Example 3: BACE1 cleaves endogenous Occludin in human umbilical vein endothelial cells (HUVEC) while promoting the degradation of other tight junction proteins.

[0176] 1. Experimental Procedure

[0177] HUVEC cell lines can produce endogenous Occludin, Claudin-1, JAMA, and ZO-1. Therefore, the inventors constructed a stable HUVEC cell line transfected with HA-BACE1 and used Western blot to detect changes in the tight junction proteins Claudin-1, JAMA, and ZO-1. They found that the levels of all three tight junction proteins were significantly decreased. Subsequently, using in vitro endothelial cell permeability assays, they found that BACE1 reduced the amount of tight junction proteins and also disrupted tight junction structure. Furthermore, using siRNA technology, they constructed a stable HUVEC cell line transfected with BACE1 and Occludin knockdown. They found that Claudin-1 was absent in this cell line, and the levels of JAMA and ZO-1 were decreased, confirming that BACE1 cleavage of Occludin protein directly affects the other three tight junction proteins and tight junction structure. Finally, immunofluorescence was used to label lysosomes and autophagosomes. We found that the lysosomal inhibitor A1 could inhibit the degradation of tight junction proteins Claudin-1 and ZO-1 caused by BACE1 cleavage of Occludin. At the same time, these two proteins co-localized with the markers EEA1 and LC3 of lysosomes and autophagosomes under confocal microscopy. Therefore, it was confirmed that BACE1 can cleave endogenous Occludin and promote the breakdown of tight junction structures and protein degradation, thereby affecting the structure and function of tight junctions.

[0178] 2. Experimental Materials

[0179] HUVEC cells and cell culture reagents were purchased from GIBICO (Carlsbad, CA) and C3 (Merck). The cell culture method was the same as in Example 1. HUVEC cells were transfected using the Effectene Transfection Reagent Kit (Qiagen) and Lipofectamine 3000 (Life Technologies) or Lipofectamine LTX (Life Technologies), Transwell (Corning, CLS3379-2EA), lysosomal inhibitor A1 (purchased from CST), lysosomal marker EEA1 (purchased from BD Biosciences), and autophagy marker LC3 (purchased from MBL).

[0180] Primary antibodies: anti-Occludin antibody (Proteintech, 13409-1-AP), anti-ZO-1 antibody (Invitrogen, 61-7300), anti-JAM-A antibody (Santa Cruz Biotechnology, sc-53624(1H2A9)), anti-Claudin-1 antibody (Proteintech, 13050-1-AP), anti-HA tag antibody (MBL, M180-3), anti-Rab7 antibody (CST, 9367S), anti-Rab5 antibody (CST, 3547S), anti-GM130 antibody (CST, 12480S), anti-LAMP1 antibody (CST, 9091), anti-LC3 antibody (MBL, M152-3), anti-EEA1 antibody (BDBiosciences, 610456).

[0181] Secondary antibodies: Alexa Fluor 568 - anti-rabbit IgG (H+L), Alexa Fluor 488 - anti-mouse IgG (H+L), Alexa Fluor 405 - anti-mouse IgG (H+L) antibody (Life Technologies); Alexa Fluor 594 - anti-rabbit IgG (H+L), Alexa Fluor 647 - anti-mouse IgG (H+L), F(ab')2 antibody (CST).

[0182] The siRNA was purchased from GenePharma, and siRNA transfection was performed using Lipofectamine RNAiMAX (Life Technologies).

[0183] OccludinsiRNA sequence:

[0184] si-Occludin: 5'-CCGAAUCAUUAUGCACCAATT-3'SEQ ID NO: 21);

[0185] si-control sequence: 5'-UUCUCCGAACGUGUCACGUTT-3' (SEQ ID NO: 22) (This can be obtained by randomly scrambling the above siRNA-Occludin oligonucleotide sequence).

[0186] 3. Specific operations

[0187] (1) HA-BACE1 and EGFP-BACE1 were obtained by using BACE1 cDNA and vectors HA-PKH3 and EGFP-N3, respectively.

[0188] (2) BACE1 cleavage of endogenous Occludin in HUVEC cells: HUVEC cells were transfected with HA-vector or HA-BACE1. The experiment was divided into four groups: HUVEC cells transfected with 0.3 μg HA-vector, 0.3 μg HA-BACE1, 0.6 μg HA-vector, and 0.6 μg HA-BACE1.

[0189] Cell samples were lysed and collected using cell lysis buffer (phosphate buffer, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, and protease inhibitor cocktail). Cells were lysed at room temperature, and then an equal volume of 2×SDS electrophoresis loading buffer was added. The samples were then boiled in water for 10 minutes to prepare SDS electrophoresis samples. Changes in Occludin-FL, Occludin-CTF, and Occludin-NTF were detected using Western blot. The immunoblotting procedure was the same as in Example 1.

[0190] (3) In vitro human umbilical vein endothelial cell permeability detection experiment: HUVECs (2×10⁻⁶) were used to detect the permeability of human umbilical vein endothelial cells. 4 (Inoculation per chamber) was performed in the upper chamber of the transwell (0.33 cm). 2 The cells were incubated in a 0.4 μm well at 37°C for 4-5 days until the culture medium no longer seeped into the lower chamber from the upper chamber. Pretreatment was performed according to experimental groups: HUVEC cells were transfected with different concentrations of HA-vector or corresponding concentrations of HA-BACE1. The experiment was divided into four groups: HUVEC cells transfected with 0.4 μg HA-vector, 0.4 μg HA-BACE1, 0.8 μg HA-vector, or 0.8 μg HA-BACE1. 10 kDa or 70 kDa of FITC-labeled dextran (diluted to 1 mg / mL with serum-free DMEM) was added to the upper chamber. The transwell was incubated in a 37°C incubator. After 30, 60, 90, and 120 minutes, 20 μl of the lower chamber was transferred to a black 96-well plate, and the absorbance of each well was measured using a microplate reader (490 nm, 520 nm).

[0191] (4) Experiment to detect the effect of si-occludin on the effect of BACE1 on other tight junction proteins: HUVEC cells were transfected with EGFP-BACE1 and siRNA was added. The experiment was divided into four groups: transfected with EGFP-vector and si-control, transfected with EGFP-BACE1 and si-control, transfected with EGFP-vector and si-occludin, and transfected with EGFP-BACE1 and si-occludin.

[0192] Cell samples were lysed and collected using cell lysis buffer (phosphate buffer, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, and protease inhibitor cocktail). Cells were lysed at room temperature, and then an equal volume of 2×SDS electrophoresis loading buffer was added. The samples were then boiled in boiling water for 10 minutes to prepare SDS electrophoresis samples.

[0193] The changes in Occludin, Claudin-1, JAMA, and ZO-1 were detected using Western blot technology. The procedure for Western blotting was the same as in Example 1.

[0194] (5) Experiment to detect the effect of lysosomal inhibitor A1 on the effect of BACE1 on other tight junction proteins: HUVEC cells were transfected with EGFP-BACE1 and lysosomal inhibitor A1 (final concentration 100 nM) were added. The experiment was divided into four groups: transfected with EGFP-vector; transfected with EGFP-vector and added with A1; transfected with EGFP-BACE1; and transfected with EGFP-BACE1 and added with A1.

[0195] Cell samples were lysed and collected using cell lysis buffer (phosphate buffer, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, and protease inhibitor cocktail). Cells were lysed at room temperature, and then an equal volume of 2×SDS electrophoresis loading buffer was added. The samples were then boiled in boiling water for 10 minutes to prepare SDS electrophoresis samples.

[0196] The changes in Occludin, Claudin-1, JAMA, and ZO-1 were detected using Western blot technology. The procedure for Western blotting was the same as in Example 1.

[0197] (6) Immunofluorescence experiment to observe the colocalization of tight junction proteins with lysosomes and autophagosomes: Cells were treated according to the experimental design. Cells were washed three times with PBS at room temperature for 5 minutes each time. Cells were fixed with 4% paraformaldehyde for 15 minutes and washed five times with PBS for 5 minutes each time. Cells were treated with 0.25% Triton-100 at room temperature for 30 minutes. Cells were blocked with 3% FBS (PBS solution) at room temperature for 1 hour. After removing the blocking solution, the primary antibody (using each tight junction antibody, anti-EEA1 antibody, and anti-LC3 antibody) was diluted according to the appropriate ratio and incubated overnight in a shaker at 4 degrees Celsius. The primary antibody was removed the next day. Cells were washed three times with PBS for 5 minutes each time. Secondary antibody was diluted according to the appropriate ratio and incubated at room temperature in the dark for 40 minutes to 1 hour. Finally, the secondary antibody solution was removed and the cells were washed five times with PBS for 5 minutes each time. After that, the colocalization of tight junction proteins with lysosomes and autophagosomes was observed under a fluorescence microscope with 63x oil immersion and images were acquired.

[0198] 4. Results Analysis

[0199] Depend on Figure 8 The results showed that BACE1 can also specifically cleave endogenous Occludin in HUVEC cells, while causing a decrease in the levels of other tight junction proteins.

[0200] Depend on Figure 9 The results showed that with the increase of BACE1 concentration, the permeability of endothelial cells induced by BACE1 increased, indicating that BACE1 can cause damage to the tight junction structure of endothelial cells.

[0201] Depend on Figure 10 The results showed that in the presence of Occludin, the concentration of other tight junction proteins decreased. However, after knocking out Occludin, BACE1 was not cleaved, and the concentration of other tight junction proteins did not decrease significantly. This indicates that BACE1 does not directly cleave other tight junction proteins, but rather causes a decrease in their levels by acting on Occludin.

[0202] Depend on Figure 11 The results showed that the decrease in tight junction proteins JAMA and Claudin-1 caused by BACE1 was inhibited after the addition of lysosomal inhibitor A1, indicating that the decrease in JAMA and Claudin-1 levels caused by BACE1 is mediated through lysosomal degradation.

[0203] Depend on Figure 12The results showed that after using the lysosomal inhibitors A1 and BACE1, the co-localization of lysosomal EEA1 and autophagosome LC3 with the tight junction membrane protein Claudin-1 was significantly increased, but no co-localization with the cytoplasmic protein ZO-1 was observed. Combined with these experimental results, it is indicated that BACE1 cleavage of Occludin causes instability in the tight junction structure, leading to the degradation of tight junction membrane proteins via the lysosomal pathway. Meanwhile, ZO-1, located in the cytoplasm, may be degraded through other pathways, thus resulting in a decrease in the content of all tight junction proteins.

[0204] Example 4: Constructing a mouse model of endothelial cells overexpressing BACE1.

[0205] 1. Experimental Procedure

[0206] Using transgenic technology, a BACE1 overexpression model mouse, VE-BACE1 (using the vector 5'LTR-Tie2 Promoter-BACE1-WRE-3'Poly A), was constructed using C57BL / 6J mice as the background. The resulting first-generation VE-BACE1 mice were continuously mated with C57BL / 6J mice from the same background to obtain progeny stably expressing high levels of BACE1 in endothelial cells. Western blot and immunofluorescence techniques were used to verify the successful construction of this animal model. VE-BACE1 mice showed reduced Occludin protein levels in the brain, along with corresponding reductions in tight junction proteins ZO-1, JAMA, and Claudin-1. Western blot and immunofluorescence techniques were used to detect changes in Occludin protein levels (ZO-1, JAMA, and Claudin-1) in VE-BACE1 mice of different ages. We found that VE-BACE1 mice had reduced Occludin protein, increased Occludin-CTF, and reduced tight junction proteins ZO-1, JAMA, and Claudin-1.

[0207] 2. Experimental Materials

[0208] VE-BACE1 mice were obtained from C57BL / 6J background mice through transgenic technology and were stably inherited after six generations of breeding.

[0209] Primary antibodies: anti-BACE1 antibody (CST, 5606S), anti-Occludin antibody (Invitrogen, 331594 (OC-3F10)), anti-ZO-1 antibody (Invitrogen, 61-7300), anti-JAM-A antibody (SantaCruz, sc-53624 (1H2A9)), anti-claudin-1 antibody (Abcam, ab15098), anti-CD31 antibody (BD Biosciences, 550274), anti-CD34 antibody (Invitrogen, 14-0341).

[0210] Secondary antibodies: Alexa Fluor 594 - Anti-rabbit IgG (H+L), F(ab')2 antibody (CST), Alexa Fluor 568 - Anti-rabbit IgG (H+L) (Life Technologies), Alexa Fluor 568 - Anti-mouse IgG (H+L) (Life Technologies), Alexa Fluor 488 - Anti-rat IgG (H+L) antibody (Abcam).

[0211] 3. Specific operations

[0212] (1) Western blot detection of BACE1 overexpression in vascular tissues of VE-BACE1 mice: 15-month-old VE-BACE1 and WT mice were anesthetized with sodium pentobarbital, perfused with 1×PBS, and the brain, heart, liver, spleen, kidneys, muscles, and peripheral tissues were completely removed using surgical instruments and stored at -80℃. A suitable amount of the above mouse tissues was taken and homogenized in cell lysis buffer (phosphate buffer, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, protease inhibitor cocktail) on ice using a Durns tissue homogenizer, sonicated for 4 minutes, and centrifuged at 160,000g for 30 minutes at 4℃.

[0213] BACE1 expression was detected using Western blot, following the same procedure as in Example 1. The results are as follows: Figure 13 As shown.

[0214] (2) Immunohistochemical detection of BACE1 overexpression in VE-BACE1 mice: 15-month-old VE-BACE1 and WT mice were treated according to experimental requirements (mouse blood vessels were labeled with CD31 antibody, a marker of vascular endothelial cells). Mice were anesthetized with sodium pentobarbital and perfused with pre-cooled 1×PBS solution. Intact brain tissue was removed using surgical instruments. After fixation with 4% PFA for 24 h, the tissue was dehydrated using a sucrose gradient and cut into 30-50 μm brain slices. Cells were treated with 0.5% Triton-100 at room temperature for 30 minutes. Cells were then blocked overnight at 4°C with 3% goat serum and 1% BSA (in PBS solution). The next day, the blocking solution was removed. Cells were then incubated overnight at 4°C with a diluted primary antibody solution. The primary antibody solution was removed the following day. Cells were washed five times with PBS for 5 minutes each time. A diluted secondary antibody solution was added, and the cells were incubated at room temperature in the dark for 40 minutes to 1 hour. Finally, the secondary antibody solution was removed, and cells were washed five times with PBS for 5 minutes each time. Images were then observed and acquired under a confocal fluorescence microscope. Figure 14 As shown, CD31 and CD34 are markers of vascular endothelial cells.

[0215] (3) Western blot analysis of tight junction protein expression levels in VE-BACE1 mice: VE-BACE1 and WT mice of different ages (9, 15, and 21 months) were anesthetized with sodium pentobarbital, perfused with 1×PBS, and their brains were completely removed using surgical instruments and stored at -80℃. A suitable amount of brain tissue from these mice was homogenized in cell lysis buffer (phosphate buffer, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, and a protease inhibitor cocktail) on ice using a Dunns tissue homogenizer, sonicated for 4 minutes, and centrifuged at 160,000g for 30 minutes at 4℃.

[0216] The expression levels of Occludin, ZO-1, JAMA, and Claudin-1 were detected using Western blot. The Western blot procedure was the same as in Example 1, and the results are as follows. Figure 15 As shown.

[0217] (4) Immunohistochemical detection of vascular tight junction protein expression levels in VE-BACE1 mice: 21-month-old VE-BACE1 and WT mice were treated according to experimental requirements. Mice were anesthetized with sodium pentobarbital and perfused with pre-cooled 1×PBS solution. Complete brain tissue was removed using surgical instruments. The cells were fixed with 4% PFA for 24 hours, dehydrated using a sucrose gradient, and cut into 30-50 μm slices. Cells were treated with 0.5% Triton-100 at room temperature for 30 minutes, blocked overnight at 4°C with 3% goat serum and 1% BSA (in PBS solution). The next day, the blocking solution was removed, and the cells were incubated overnight at 4°C with a diluted primary antibody solution. The primary antibody was removed the next day; cells were washed 5 times with PBS for 5 minutes each time; a diluted secondary antibody solution was added, and the cells were incubated at room temperature in the dark for 40 minutes to 1 hour. Finally, the secondary antibody solution was removed, and cells were washed 5 times with PBS for 5 minutes each time. Images were then observed and acquired under a confocal fluorescence microscope. Figure 16 As shown.

[0218] 4. Results Analysis

[0219] Depend on Figure 13 and 14 The results showed that the expression level of BACE1 in the tissues of VE-BACE1 mice was significantly increased, and immunohistochemical experiments showed that BACE1 and vascular endothelial cells were completely co-localized, indicating that the VE endothelial cell overexpression BACE1 model mouse was successfully constructed.

[0220] Depend on Figure 15 and 16 The results showed that, compared with wild-type mice of the same age, the levels of tight junction proteins Occludin, Claudin-1, JAMA and ZO-1 in the brains of VE-BACE1 mice were decreased.

[0221] Example 5: VE-BACE1 mice showed damage to brain blood vessels and neuron-vascular unit (NVU).

[0222] 1. Experimental Procedure

[0223] Immunohistochemistry was used to label blood vessels, along with the CD31 antibody (a marker of vascular endothelial cells) and vascular morphology. We found a significant reduction in vascular endothelial staining in the cerebral cortex, striatum, and hippocampus of 21-month-old VE-BACE1 mice, indicating vascular endothelial damage and loss in these mice (see [link to article]). Figure 17 Furthermore, immunohistochemistry was used to detect the expression level of caveolin-1 (the protein level increases in cases of vascular endothelial injury). We found that the expression level of caveolin-1 was significantly increased in the cerebral cortex of VE-BACE1 mice, further confirming vascular endothelial injury (see...). Figure 18Simultaneously, immunohistochemistry was used to examine changes in astrocytes and pericytes within the NVU structure. First, AQP4 antibody was used to label changes in the terminal appendages of astrocytes. We found that AQP4 protein levels were significantly decreased in the VE-BACE1 cortex, striatum, and hippocampus (see...). Figure 19 Secondly, using Desmin protein antibody to label pericytes, we found that Desmin protein levels were significantly decreased in VE-BACE1 brain cells, indicating damage to NVU structure (see...). Figure 20 ).

[0224] 2. Experimental Materials

[0225] Transgenic mice that were stably inherited after six generations of breeding were selected from VE-BACE1 mice.

[0226] Primary antibodies: anti-CD31 antibody (BD Biosciences, 550274), anti-CD34 antibody (Invitrogen, 14-0341), anti-AQP4 antibody (Proteintech, 16473-1-AP), anti-Desmin antibody (Abcam, ab32362), anti-Caveolin-1 antibody (CST, 3267S).

[0227] Secondary antibodies: Alexa Fluor 594 - Anti-rabbit IgG (H+L), F(ab')2 antibody (CST), Alexa Fluor 568 - Anti-rabbit IgG (H+L) (Life Technologies), Alexa Fluor 568 - Anti-mouse IgG (H+L) (Life Technologies), Alexa Fluor 488 - Anti-rat IgG (H+L) antibody (Abcam).

[0228] 3. Specific operations

[0229] Immunohistochemical labeling of VE-BACE1 mouse brain blood vessels: The specific operation is the same as in Example 4.

[0230] 4. Results Analysis

[0231] The above experimental results ( Figures 17-20 This study showed that VE-BACE1 mice experienced loss and damage to the vascular endothelial structure in the brain, and that the neuronal-vascular unit's constituent structures—the terminal foot of astrocytes and pericytes—also showed significant reduction and damage.

[0232] Example 6: Significant degeneration of capillaries and venules in the brain of VE-BACE1 mice.

[0233] 1. Experimental Procedure

[0234] Immunohistochemistry was used to label different vascular types in the brains of 21-month-old VE-BACE1 mice, including arteries, veins, and capillaries, to observe the damage to different types of blood vessels. First, all blood vessels in the VE-BACE1 brain were labeled with a vascular basement membrane marker, laminin antibody, and then with a parietal cell marker, PDGFRβ antibody. We found that the morphology and number of large blood vessels in VE-BACE1 mice were not significantly different from those in WT mice, while the number of small blood vessels in the brains of VE-BACE1 mice was significantly reduced. Figures 21-22 Furthermore, we labeled cerebral arteries with SMA protein antibody (a marker of vascular smooth muscle cells), and labeled cerebral blood vessels in VE-BACE1 mice with vWF protein antibody (vWF protein is expressed in arteries and venules), MCT1 protein antibody (MCT1 protein is expressed in venules and capillaries), and VCAM-1 protein antibody (VCAM-1 protein is mainly expressed in veins). We found a significant reduction in capillaries and venules in the VE-BACE1 brain. This indicates significant degeneration of capillaries and venules in the VE-BACE1 mouse brain. Figures 23-26 ).

[0235] 2. Experimental Materials

[0236] Primary antibodies: anti-Tagln antibody (Proteintech, 10493-1-AP), anti-vWF (Proteintech, 11778-1-AP), anti-VCAM-1 (CST, D8U5V), anti-SMA (Abcam, ab5694), anti-Laminin antibody (Sigma-Aldrich, L9393), anti-MCT1 antibody (Proteintech, 20139-1-AP), anti-PDGFRβ antibody (BD Biosciences, 558821);

[0237] Secondary antibodies: Alexa Fluor 594 - Anti-rabbit IgG (H+L), F(ab')2 antibody (CST), Alexa Fluor 568 - Anti-rabbit IgG (H+L) (Life Technologies), Alexa Fluor 568 - Anti-mouse IgG (H+L) (Life Technologies), Alexa Fluor 488 - Anti-rat IgG (H+L) antibody (Abcam).

[0238] 3. Specific operations

[0239] (1) Immunohistochemical labeling of various blood vessels in the brain of VE-BACE1 mice: The specific operation is the same as in Example 4.

[0240] 4. Results Analysis

[0241] The above experimental results ( Figures 21-26 The results showed that, compared with WT mice, VE-BACE1 mice did not show significant changes in large blood vessels (arteries and veins) in the brain, but the number of small blood vessels (capillaries and venules) was significantly reduced and damaged, indicating that cerebrovascular damage in VE-BACE1 mice was mainly concentrated in small blood vessels.

[0242] Example 7: Blood-brain barrier damage in VE-BACE1 mice.

[0243] 1. Experimental Procedure

[0244] Immunohistochemistry and Western blot analysis revealed a significant increase in fibrin(ogen) levels in the brains of VE-BACE1 mice after perfusion. This indicates damage to the blood-brain barrier in VE-BACE1 mice, allowing fibrin(ogen) to cross the blood-brain barrier and enter the brain (see [link to article]). Figure 27 Simultaneously, the blood-brain barrier permeability was detected by intravenous injection of the fluorescent dye Alexa Fluor 555-conjugated cadaverine into the orbital vein of VE-BACE1 mice. Fluorescence detection of VE-BACE1 brain slices and brain homogenates showed that the blood-brain barrier permeability of VE-BACE1 mice was significantly increased, further indicating that VE-BACE1 mice suffered from blood-brain barrier damage. Figure 28 Furthermore, we found that VE-BACE1 mice exhibited pathological manifestations caused by blood-brain barrier damage, including intracranial inflammation, synaptic structural damage, intracranial microbleeds, and impaired spatial learning and memory abilities. Immunohistochemistry was used to detect the distribution and number of microglia and astrocytes, and we found that microglia (labeled with iba-1)... Figure 30 ) and astrocytes (labeled by GFAP) Figure 29 The increased number of neurons around blood vessels indicates that impaired blood-brain barrier function in VE-BACE1 mice promotes inflammatory responses. Secondly, immunohistochemical staining was used to label neurons (NeuN antibody-labeled neurons). Figure 31 ), using SNAP25 protein antibody (presynaptic marker) ( Figure 32 ) and PSD95 protein antibody (postsynaptic marker) Figure 33The distribution of presynaptic and postsynaptic structures was marked. We found no significant change in the number of neurons in VE-BACE1 mice (NeuN staining), but a reduction in synaptic structures (SNAP25 and PSD95 staining), indicating synaptic damage in VE-BACE1 mice. Using Prussian blue staining to detect intracranial microbleeds in VE-BACE1 mice at 15 and 21 months, we found obvious microbleed symptoms in the brains of VE-BACE1 mice. Figures 34-35 Finally, the Barnes maze behavioral test was used to examine the spatial learning and memory abilities of VE-BACE1 mice. We found that VE-BACE1 mice exhibited impaired spatial learning and memory functions. Figure 36 ).

[0245] 2. Experimental Materials

[0246] Primary antibodies: anti-fibrinogen antibody (Abcam, ab34269), anti-tubulin antibody (Proteintech, 66031-1-lg), anti-GFAP antibody (Millipore, IF03L), anti-Iba-1 antibody (Wako, 019-19741), anti-NeuN antibody (Millipore, MAB377), anti-SNAP25 antibody (Abcam, ab41455), anti-PSD95 antibody (CST, 3450S), cadaverine-Alexa Fluor 555 (Life Technologies, A30677), and Rhus blue staining kit (Solarbio, Cat#G1424).

[0247] Secondary antibodies: Alexa Fluor 594 - Anti-rabbit IgG (H+L), F(ab')2 antibody (CST), Alexa Fluor 568 - Anti-rabbit IgG (H+L) (Life Technologies), Alexa Fluor 568 - Anti-mouse IgG (H+L) (Life Technologies), Alexa Fluor 488 - Anti-rat IgG (H+L) antibody (Abcam).

[0248] 3. Specific operations

[0249] (1) Western blot analysis of fibrinogen content in the brain: VE-BACE1 mice and their corresponding WT mice were treated according to experimental requirements. After anesthetizing with sodium pentobarbital, the mice were perfused with pre-cooled 1×PBS solution. Intact brain tissue was removed using surgical instruments and stored at -80℃. Tissue lysis buffer (1% Nonidet P-40, 0.5% sodium deoxycholate, 0.5% SDS, and protease inhibitor cocktail (Roche)) was added to the whole brain tissue at a 1:30 volume ratio. The tissue was homogenized into uniform microparticles in a homogenizer, the supernatant was discarded, and 3M urea solution was added at a 1:40 volume ratio. The mixture was then homogenized in a Durns tissue homogenizer and stirred at 37℃ for 2 hours. After centrifugation at 14000xg for 30 minutes, the supernatant was discarded, and 3M urea solution was added at a 1:40 volume ratio. The mixture was then homogenized in a homogenizer and stirred at 65℃ for 30 minutes to obtain a sample suitable for Western blot analysis. The procedure for Western blotting is the same as in Example 1.

[0250] (2) Immunohistochemical test to detect the content of fibrin(ogen) in the brain: The immunohistochemical experiment is the same as in Example 4.

[0251] (3) Blood-brain barrier permeability experiment: VE-BACE1 and WT mice were anesthetized with sodium pentobarbital according to experimental requirements. After anesthesia, the mice were injected with 0.5 mg / ml cadaverine-Alexa Fluor 555 tracer into their orbits. The injection volume was calculated based on the body weight of each mouse at 25 μg / g. After 2 hours of circulation, the mice were perfused with 1×PBS for 5 minutes. The brain tissue was then completely removed using surgical instruments and divided into two hemispheres. One hemisphere was weighed and added to 1% Triton X-100 in 1×PBS solution. It was homogenized on ice and then centrifuged at 21000xg for 20 minutes at 4°C. The supernatant was collected into a black 96-well plate, and the absorbance of the liquid in each well was measured using a microplate reader (542 nm, 572 nm). The other hemisphere was fixed with 4% PFA for 24 hours, dehydrated with a sucrose gradient, and cut into 30-50 μm brain slices. The slices were then observed and images were acquired under a 20x microscope using a tissue FACXS system (Tissue Gnostics).

[0252] (4) Prussian blue staining experiment: VE-BACE1 mice and their corresponding WT mice at 15 and 21 months of age were treated according to experimental requirements. Mice were anesthetized with sodium pentobarbital and perfused with pre-cooled 1×PBS solution. Intact brain tissue was removed using surgical instruments and stored at -80℃. One hemisphere of the brain was fixed with 4% PFA for 24 h, dehydrated using a sucrose gradient, and then sagittally sectioned into 14-18 μm brain slices (180 slices per hemisphere). From the lateral to the medial side, one slice was randomly selected from every three slices, yielding 60 slices per hemisphere. Staining was performed with neutral red for 2 minutes, followed by Prussian blue staining in a 1:1 solution of 20% hydrochloric acid and 10% potassium cyanide. After staining, the slices were dehydrated with a series of alcohols, washed with xylene, mounted with neutral lipids, and observed and imaged at 20x magnification using a tissue FACXS system. The area occupied by blue staining spots in the brain slices was quantified using the ImageJ image processing system.

[0253] (5) Barnes Maze Experiment: The Barnes maze consists of a white circular platform (91 cm in diameter) 80 cm above the ground, with 20 equally spaced holes (5 cm in diameter) along the circumference. A black escape tunnel is placed below the target hole. Four bright lights are placed above the platform as a weak aversive stimulus, allowing mice to use spatial cues placed around the room to locate the target hole. VE-BACE1 and WT mice of different ages were selected for the following experiment.

[0254] 1) The day before the experiment, the animals were individually placed from the target hole into the target box for 3 minutes to acclimatize;

[0255] 2) Experimental Phase: Place the animal in a plastic cylinder in the center of the maze and restrict its movement for 5 seconds. Remove the cylinder and start the timer. If all four limbs of the animal enter the target box, it is considered to have correctly found the target hole, and the animal stays in the box for 30 seconds. Observe each animal for 3 minutes at a time. If the animal still cannot find the target hole during this period, remove the animal from the maze, place it in the target box, and let it stay for 30 seconds. Train four times a day for four consecutive days.

[0256] Starting with the second training session, the maze was randomly rotated one or more times before each training session, but the target box remained in the same position. This was done to prevent the animals from relying on smell rather than memory to locate the target hole. The experiment recorded the following parameters: time to reach the target hole.

[0257] 3) On days 5 and 12, a 90-second targeted experiment was conducted to examine the mice's spatial and long-term memory. The following parameters were recorded: time to reach the target hole.

[0258] 4. Results Analysis

[0259] Depend on Figure 27 and28 The results showed that the fibrin(ogen) content in the brain of VE-BACE1 mice was significantly higher than that of WT mice, indicating that the blood-brain barrier of VE-BACE1 mice was damaged, allowing fibrin(ogen) to cross the blood-brain barrier and enter the brain.

[0260] Depend on Figure 29 and 30 The results showed that perivascular astrocytes (labeled by GFAP) in the brains of VE-BACE1 mice ( Figure 29 ) and microglia (labeled by iba-1) Figure 30 The level of VE-BACE1 mice was significantly higher than that in WT mice, indicating that VE-BACE1 mice had an inflammatory response around blood vessels in the brain.

[0261] Depend on Figures 31-33 The results showed that the number of neurons in VE-BACE1 mice was not significantly different from that in WT mice, but the staining results of synaptic structure in VE-BACE1 mice showed a reduction, indicating that VE-BACE1 mice suffered synaptic damage.

[0262] Depend on Figure 34 The results showed that the amount of cadaverine, a fluorescent dye, that permeated across the blood-brain barrier in VE-BACE1 mice was significantly higher than that in WT mice, and this difference became more pronounced with age. This indicates that the blood-brain barrier permeability of VE-BACE1 mice is increased, and the blood-brain barrier is damaged, with the damage becoming more pronounced with age.

[0263] Depend on Figure 35 The results showed that VE-BACE1 mice had significantly higher rates of hemorrhage in various brain regions compared to WT mice, and this difference became more pronounced with age, indicating that VE-BACE1 mice exhibited pathological features of intracranial hemorrhage, which became more pronounced with age.

[0264] Depend on Figure 36 The results showed that VE-BACE1 mice exhibited a decline in spatial learning ability, as well as short-term and long-term memory functions, compared to WT mice, indicating that VE-BACE1 mice suffered from impairment in spatial learning ability and memory function.

[0265] Example 8: BACE1 inhibitors can improve learning and memory impairment in VE-BACE1 mice.

[0266] 1. Experimental Procedure

[0267] Multiple behavioral experiments were used to examine whether the learning and memory abilities of VE-BACE1 mice improved after treatment with the BACE1 inhibitor MK-8931. We found that VE-BACE1 mice treated with MK-8931 at a dose of 1.5 mg / kg for 2 months showed significantly improved learning and memory abilities in the Barnes maze, new object recognition, and Two-Trial Y-maze tests compared to the control group of untreated VE-BACE1 mice. Furthermore, the difference between these two groups was smaller compared to the control group (WT mice). This indicates that BACE1 inhibitors can improve learning and memory impairment in VE-BACE1 mice and may be a promising new drug for the treatment of CSVD.

[0268] 2. Experimental Materials

[0269] BACE1 inhibitors MK-8931 (Merck), DMSO (Thermo).

[0270] 3. Specific operations

[0271] (1) Treatment of mice: 15-month-old littermate WT mice and VE-BACE1 mice were divided into two groups. One group was given 1.5 mg / kg body weight of BACE1 inhibitor MK-8931 by drinking water, and the other group was given the same dose and administration method of DMSO by drinking water. The treatment was continued for 2 months.

[0272] (2) Open field test: This is a method to evaluate the autonomous behavior, exploratory behavior and stress level of experimental animals in a new open environment. Mice will have fear of new open environments, so they mainly move in the peripheral area and less in the central area. However, the exploratory nature of mice will motivate them to move in the central area. Therefore, the movement trajectory and anxiety of mice can be observed during their activity.

[0273] 1) Experimental Stage: The inner walls of the open field reaction chamber were painted white, and an infrared and digital camera was mounted directly above it, its field of view covering the entire open field. The lighting in the open field was artificially set to simulate "nighttime" for the mice's activity, with a light source on one wall simulating moonlight. Experimenters and equipment, including computers, were located in another room to minimize interference with the animals. The bottom of the open field reaction chamber was a 50×50cm square. Before the experiment, computer software divided the bottom into 25 equally shaped small squares, including 12 outer squares and 13 inner squares. After setup, the mice were placed facing the wall in one of the four corner squares and allowed to freely explore the environment for 5 minutes. The computer software then analyzed the total exploration distance of the mice and the total exploration time within each square.

[0274] 2) Analysis phase: The computer software automatically analyzes the movement distance of each mouse within 5 minutes. The value can be used to assess the mouse's motor ability. The greater the movement distance, the stronger the mouse's motor ability. The computer software automatically analyzes the exploration time in each square. The experimenters calculate the total exploration time in the 12 surrounding squares and the total time spent in the 13 inner squares. The anxiety of the mice is assessed using the formula: Total exploration time in the 12 surrounding squares / (Total exploration time in the 12 surrounding squares + Total time spent in the 13 inner squares). A higher ratio indicates that the mouse is more anxious.

[0275] (3) Spontaneous Y-maze experiment: The Y-maze consists of 3 arms of equal length (50cm×18cm×35cm), with an angle of 120 degrees between each pair of arms. There is a movable partition in the center of each arm. The inner arms and bottom of the maze are painted white.

[0276] 1) Experimental phase: Fix one of the arms as the starting arm, and place the mouse at the end of the starting arm each time to explore freely for 5 minutes. Record the order in which the mouse explores the three long arms using a video recording device.

[0277] 2) Analysis Phase: Every three exploration sequences are considered as one response count. Explorations without duplicate arms are counted as one correct response; otherwise, they are considered one incorrect response. Calculate the number of correct responses / (number of correct responses + number of incorrect responses) * 100%. The higher the percentage, the stronger the mouse's memory ability.

[0278] (4) Barnes Maze Experiment: The operation method is the same as in Example 7.

[0279] (5) New Object Recognition Experiment: A learning and memory test method established based on the principle that mice have an innate tendency to explore new objects. The device parameters are a square with a base of 50×50cm, silver-gray color, and a height of 40cm on all four sides.

[0280] 1) The mice participating in the experiment were placed in the device for 10 minutes at a fixed time each day for a total of 3 days to adapt;

[0281] 2) Experimental Phase: Two identical objects were placed at opposite ends of a side wall. A mouse was placed in the testing area with its back to both objects, ensuring the distance from the mouse's nose to each object was equidistant. Recording was initiated before the mouse was placed in the room, and the experimenter immediately left the testing room after the mouse was placed. This process lasted 10 minutes. Two hours later, one of the old objects was replaced with a new object of equal height and volume but a different shape. The mouse was again placed with its back to both objects, its nose equidistant from the objects, and observed for 5 minutes, recording the mouse's contact with the two objects.

[0282] 3) Analysis phase: Record the time each mouse spends exploring the new and old objects, and calculate the new object exploration time / (new object exploration time + old object exploration time) * 100%. The higher the percentage, the stronger the mouse's memory ability.

[0283] (6) Western blot analysis of the effect of BACE1 inhibitor MK-8931 on the level of tight junction protein in brain blood vessels: After all behavioral tests were completed, all 18-month-old VE-BACE1 and WT mice participating in the drug administration experiment were anesthetized with sodium pentobarbital, perfused with 1×PBS, and the brain, heart, liver, spleen, kidney, muscle, and peripheral tissues were completely removed by surgical instruments and quickly stored at -80℃. An appropriate amount of the left brain tissue of the above mice was placed in cell lysis buffer (phosphate buffer, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, protease inhibitor cocktail), homogenized on ice using a Durns tissue homogenizer, sonicated for 4 minutes, and centrifuged at 160,000g for 30 minutes at 4℃.

[0284] BACE1 expression was detected using Western blot, and the procedure for Western blot was the same as in Example 1.

[0285] 4. Results Analysis

[0286] From the appendix Figures 37-42 The results showed that chronic low-dose MK-8931 treatment significantly improved the loss of cerebral vascular tight junction proteins, especially Occludin, JAM-A, and Claudin-1, in VE-BACE1 mice. Behavioral studies showed that... Figure 43 and 44 The results showed that the BACE1 inhibitor MK-8931 had no restorative effect on motor damage in VE-BACE1 mice, nor did it affect the mice's anxiety. Figures 45-48 The results showed that the memory ability of VE-BACE1 mice was significantly improved after treatment with the BACE1 inhibitor MK-8931.

[0287] In summary, BACE1 can specifically cleave the tight junction protein Occludin and cause the degradation of other tight junction proteins, JAMA, Claudin, and ZO-1, ultimately leading to the disruption of endothelial cell tight junctions. Based on this finding, a mouse model of vascular endothelial cell-specific overexpression of BACE1 was constructed. In this mouse model, it was verified that VE-BACE1 can cause a decrease in tight junction proteins and disruption of tight junction structures in vascular endothelial cells, leading to vascular structural damage and blood-brain barrier injury, ultimately causing intracranial inflammation, damage to intracranial NVUs, cerebral hemorrhage, and a decline in spatial learning and memory abilities in mice. Therefore, BACE1 can serve as a novel therapeutic target for diseases related to small vessel injury in the brain.

[0288] It should be noted that the embodiments described above are only for explaining the present invention and do not constitute any limitation on the present invention. The present invention has been described with reference to typical embodiments, but it should be understood that the words used therein are descriptive and explanatory terms, not limiting terms. Modifications can be made to the present invention within the scope of the claims, and revisions can be made to the present invention without departing from the scope and spirit of the present invention. Although the present invention described herein relates to specific methods, materials, and embodiments, it does not mean that the present invention is limited to the specific examples disclosed herein; on the contrary, the present invention can be extended to all other methods and applications with the same function.

Claims

1. Use of substances that reduce the expression level of the β-secretase (BACE1) gene and / or inhibitors of BACE1 protein activity in the preparation of drugs for the treatment or relief of diseases related to Occludin structural damage; The disease associated with the disruption of the Occludin structure is cerebral small vessel disease (CSVD). The enzyme activity inhibitor of the BACE1 protein mentioned above is C3 (CAS 797035-11-1) or MK-8931. The substance that reduces BACE1 gene expression includes small interfering RNA that promotes the degradation of BACE1 messenger RNA. The small interfering RNA is a siRNA fragment or a CRISPR gRNA that promotes the degradation of BACE1 messenger RNA, wherein the siRNA fragment is shown in SEQ ID NO: 4.

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