Application of APOE3-L159P mutation in treatment and prevention of Alzheimer disease

Abstract

The invention relates to the technical field of prevention and treatment of Alzheimer's disease, in particular to application of APOE3-L159P mutation in treatment and prevention of Alzheimer's disease. The invention relates to an application of APOE3-L159P mutation in treatment and prevention of Alzheimer's disease. According to the application method, APOE L159P mutation is overexpressed through the 2 / 8 type serotype AAV virus, and the Alzheimer's disease is prevented or the pathological manifestation of the Alzheimer's disease is relieved. Researches find that the risk and pathological characteristics of AD can be reduced by APOE3-L159P mutation, that is, AD can be treated or prevented by inducing the APOE3-L159P mutation, and a new research direction and a potential intervention target are provided for AD treatment.

Description

Technical Field

[0001] The present invention relates to the technical field of Alzheimer's disease prevention and treatment, and in particular to the application of APOE3-L159P mutation in the treatment and prevention of Alzheimer's disease. Background Art

[0002] Alzheimer's disease (AD) is a neurodegenerative disease characterized by memory loss, cognitive dysfunction, and behavioral abnormalities. Its pathological features include the aggregation of amyloid-β (Aβ) into amyloid plaques and the aggregation of hyperphosphorylated tau protein into neurofibrillary tangles, accompanied by neuronal loss and glial cell activation. Apolipoprotein E (APOE) plays an important role in the pathogenesis of AD, and different APOE isoforms play different roles.

[0003] APOE allele combinations produce three homozygous (APOE2 / 2, APOE3 / 3 and APOE4 / 4) and three heterozygous (APOE2 / 3, APOE2 / 4 and APOE3 / 4) genotypes. Different amino acid polymorphisms between alleles change the structure and function of apoE, confer different receptor and lipid binding abilities, resulting in different clearance efficiencies of lipids, inconsistent correlations with the occurrence of diseases, and provide a basis for explaining the physiological role of apoE subtypes in AD.

[0004] In recent years, several rare variants of ApoE have received much attention due to their unique association with AD, which provides new insights into the protective effects of APOE. Studies have found that several rare APOE variants have been shown to be protective against AD, such as the APOE3-V236E variant, the APOE3-Christchurch variant, and more.

[0005] The present invention aims to provide the involvement of APOE3-L159P (159 on APOE exon 3) mutation in AD progression in 5AEKO mice, as well as the protective effect on Alzheimer's disease (AD), reducing the risk and pathological characteristics of AD. Summary of the invention

[0006] The purpose of the present invention is to provide an application of the APOE3-L159P mutation in the treatment and prevention of Alzheimer's disease. AD can be treated or prevented by inducing the APOE3-L159P mutation, which provides a new research direction and potential intervention target for the treatment of AD.

[0007] In order to achieve the above object, the present invention adopts the following technical solutions:

[0008] The present invention provides application of APOE3-L159P mutation in the treatment and prevention of Alzheimer's disease.

[0009] Furthermore, the application method is:

[0010] Overexpression of the APOE L159P mutation via serotype 2 / 8 AAV virus can prevent Alzheimer's disease or alleviate the pathological manifestations of Alzheimer's disease.

[0011] The present invention has at least the following beneficial effects:

[0012] The present invention has found through research that the APOE3-L159P mutation can reduce the risk and pathological characteristics of AD, that is, AD can be treated or prevented by inducing the APOE3-L159P mutation, which provides a new research direction and potential intervention target for the treatment of AD. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings required for use in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other accompanying drawings can be obtained based on these accompanying drawings without paying any creative work.

[0014] Figure 1 The pathological manifestations of Aβ peripheral neuron damage in mice in the CN group, E3 group, and E3-L159P group;

[0015] Figure 2 Comparison of pathological manifestations of the aggregation of astrocytes and microglia around plaques in mice in the CN group, E3 group, and E3-L159P group;

[0016] Figure 3 This is the comprehensive statistical result diagram of CN group, E3 group and E3-L159P group. DETAILED DESCRIPTION

[0017] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0018] The main purpose of the present invention is to provide the APOE3-L159P mutation in 5AEKO mice to participate in the AD process, as well as the protective effect on Alzheimer's disease (AD), reduce the risk and pathological characteristics of AD, which will provide new theoretical and practical basis for the treatment of AD, as follows:

[0019] 1. Experimental plan:

[0020] By stereotaxic injection into the bilateral lateral ventricles of 5AEKO mice born on days 0-2 (P0 mice), the APOE L159P mutation was overexpressed by 2 / 8 serotype AAV virus, and brain tissue was extracted at 4.5 months of age. Neurodegenerative lesions and related changes were observed through corresponding experiments.

[0021] Experimental subjects and groups:

[0022] The experimental subjects were 5XFAD / APOE knockout mice (5AEKO), which were divided into three groups. CN group: injected with rAAV-GFAP-EGFP-WPRE-hGH pA; E3-L159P group: injected with rAAV-GFAP-APOE3(L159P)-2A-EGFP-3flag-WPRE-hGHPa; E3 group: injected with rAAV-GFAP-APOE3-2A-EGFP-3flag-WPRE-hGHPa. There were 15 female mice and male mice in each group.

[0023] 3. Experimental methods:

[0024] (1) Stereotactic brain injection: Virus injection is performed on mice on day 0-2 after birth to overexpress the corresponding mutation in the brain, successfully creating the desired mouse model.

[0025] ①Preparation before the experiment:

[0026] The genotype of the newborn mice was identified on their tails, and mice with 5A genotype were selected for injection.

[0027] ②Cleaning the needle:

[0028] The microsyringe was cleaned with alcohol and ddH2O respectively, and then the virus was drawn up.

[0029] ③Injection of mice:

[0030] Find the stereotaxic point of the brain and perform bilateral brain injection. After the injection, put the mouse back into the cage.

[0031] (2) Mouse brain tissue extraction

[0032] ① Infusion

[0033] 1) Anesthesia: 5% chloral hydrate was injected into the mouse at a ratio of 100 ul / g by intraperitoneal injection. After the mouse's respiratory rate stabilized, the limbs were fixed, the chest cavity was cut open to expose the heart, and a needle that had been rinsed with 0.5 M EDTA was pointed at the apex of the heart to draw apical blood. The blood was centrifuged at 5,500 g for 10 minutes at 4°C, and the supernatant was obtained as a plasma sample, which was stored at -80°C for later use.

[0034] 2) Use a peristaltic pump to perfuse pre-cooled phosphate buffered saline (PBS) at a flow rate of 11 ml / min for 3 minutes until the liver turns white, then stop the perfusion.

[0035] ② Tissue fixation and dehydration

[0036] 1) Carefully remove the mouse brain with forceps and place it on an ice plate. The left half of the brain is placed in pre-cooled 4% paraformaldehyde (PFA) at 4°C overnight. The other half is peeled off according to the location of different brain regions, such as the anterior and posterior cortex, hippocampus, cerebellum, thalamus, etc., and immediately frozen at -80°C for later use.

[0037] 2) The left brain was treated with 4% PFA at 4°C overnight and then transferred to 25% sucrose overnight. The next day, the brain was transferred to 30% sucrose until it was completely precipitated. The slices were cut using a Leica slicer and placed in a pre-prepared freezing solution at -20°C for later use.

[0038] refer to Figure 1-Figure 3 , Figure 1 The pathological manifestations of Aβ peripheral neuron damage in mice in the CN group, E3 group, and E3-L159P group; Figure 2 Comparison of pathological manifestations of the aggregation of astrocytes and microglia around plaques in mice in the CN group, E3 group, and E3-L159P group; Figure 3 This is the statistical result of the above two groups of figures. It can be seen that in the L159P group and the other two groups, the deposition of amyloid-β plaques decreased, alleviating the pathological manifestations of AD.

[0039] Immunofluorescence was used to verify the method of reducing AD pathological manifestations. The specific steps are as follows:

[0040] 1. Immunofluorescence

[0041] ① Use a flat-push freezing microtome to slice each piece into 50um thickness, place in freezing solution, and store at -20℃.

[0042] ② Pick out the required brain slices and place them in a twelve-well plate containing PBS to rewarm for 10 minutes, then wash them with PBS three times, 5 minutes each time.

[0043] ③ Add 2 ml of pre-prepared permeabilization solution (PBS + 0.25% Triton X-100) to each well and incubate on a shaker at room temperature for 20 minutes.

[0044] ④ Wash with PBS three times for five minutes each time.

[0045] ⑤ Perform X-34 staining and incubate the prepared X-34 staining solution (40% ETOH, 60% PBS, 1:500 10NNaOH, 1:5,000X-34) at room temperature for 20 minutes.

[0046] ⑥ Wash 3 times with 40% ETOH + 60% PBS, 2 minutes each time.

[0047] ⑦ Wash with PBS 3 times for 5 minutes each time.

[0048] ⑧ Add 2 ml of blocking solution (5% BSA) to each well and block at room temperature for 1 hour.

[0049] ⑨ Dilute the corresponding primary antibody with blocking solution and incubate the brain slices in a 48-well plate at 4°C overnight.

[0050] ⑩The next day, pick out the brain slices incubated overnight and wash them three times with PBS for 5 minutes each time.

[0051] The following operations were all protected from light. The corresponding secondary antibody was used to incubate in a 48-well plate at room temperature for 1 hour, then washed three times. The brain slices were picked up on the slide in PBS and naturally air-dried.

[0052] After the brain slices were completely air-dried, they were washed three times in dd water and then air-dried naturally.

[0053] Using Thermo Fisher's ProLong TM Gold was used for sealing.

[0054] 2. Laser spinning disk confocal imaging

[0055] All fluorescent staining of microglia and astrocytes in this section were photographed by Olympus laser spinning disk confocal.

[0056] All fluorescent slides were scanned within one week using a VS200 whole slide scanner or laser spinning disk confocal.

[0057] Before analyzing the data, a normality test was performed. Statistical analysis was performed using Student's t test and one-way analysis of variance (ANOVA). If the data did not meet the homogeneity of variance requirement, the Welch correction was used for testing. All statistics were two-tailed tests, and the experimental data were expressed as mean ± standard error (mean ± SEM). Immunofluorescence statistics were performed using Image J software, and statistical analysis and graph drawing were performed using GraphPad Prism 9.

[0058] pass Figure 1-Figure 3The results showed that the pathological manifestations of the E3-L159P group were alleviated compared with the other two groups. This indicates that the APOE3-L159P mutation can reduce the risk and pathological characteristics of AD, that is, AD can be treated or prevented by inducing the APOE3-L159P mutation.

[0059] The above shows and describes the basic principles, main features and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The above embodiments and descriptions only describe the principles of the present invention. The present invention may be subject to various changes and improvements without departing from the spirit and scope of the present invention. These changes and improvements fall within the scope of the present invention. The scope of protection claimed by the present invention is defined by the attached claims and their equivalents.

Claims

1. Application of APOE3-L159P mutation in the treatment and prevention of Alzheimer's disease.

2. The use according to claim 1, characterized in that: The application method is: Overexpression of the APOE L159P mutation via serotype 2 / 8 AAV virus can prevent Alzheimer's disease or alleviate the pathological manifestations of Alzheimer's disease.

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