Construction method and application of mouse model with cognitive impairment of Parkinson's disease

Abstract

The invention discloses a construction method and application of a mouse model with cognitive impairment of Parkinson's disease. According to the invention, after a TREM2 knockout mouse is obtained by using a gene editing technology, the TREM2 knockout mouse and an A53T alpha-Syn mouse are mated and screened, so that a Parkinson's disease cognitive impairment model mouse with a genotype of TREM2- / - / A53T alpha-Syn is obtained. The mouse obtained by the invention shows pathological deposition of hippocampus alpha-Syn, and has obvious cognitive impairment characteristics. The invention aims to establish a novel animal model for the cognitive impairment of the Parkinson's disease, and provides important help for research on occurrence, development and treatment of the cognitive impairment of the Parkinson's disease in the future.

Description

Technical Field

[0001] The present invention relates to the field of animal models, and in particular to a method for constructing and applying a mouse model of Parkinson's disease cognitive impairment. Background Art

[0002] Parkinson's disease (PD) is a common neurodegenerative disease, and cognitive dysfunction is one of the more common non-motor symptoms of PD, including Parkinson's disease mild cognitive impairment (PD-MCI) and Parkinson's disease dementia (PDD). [1] . PD-MCI has a high incidence rate and can appear in the early stages of Parkinson's disease. The annual probability of progression to dementia is 6% to 15%, which seriously affects the patient's social function and quality of life, and imposes a huge burden on patients and society. Therefore, research on cognitive impairment in Parkinson's disease is of great significance for discovering the mechanism of cognitive impairment and improving and treating cognitive impairment. Due to ethical restrictions, animal disease models are widely used in the study of Parkinson's disease, which are mainly divided into neurotoxin models and gene models. [2] However, previous model construction mainly focused on dopaminergic neuron degeneration, Lewy body formation, and motor defects, and lacked methods for constructing animal models of Parkinson's disease accompanied by cognitive impairment.

[0003] The SNCA gene was first discovered to be associated with Parkinson's disease in 1997. [3] The α-Syn encoded by it is a soluble protein mainly expressed in the presynaptic and perinuclear regions of the central nervous system. It is an important component of the Lewy bodies, a characteristic pathological sign of Parkinson's disease. In order to study the progression of Parkinson's disease, a large number of studies have focused on α-Syn transgenic animal models. Currently, three fully penetrant autosomal dominant SNCA point mutations have been identified: A53T, A30P, and E46K. [4,5] These mutations can lead to abnormal expression of α-Syn and its aggregation in neurons to form Lewy bodies, which can cause neuronal dysfunction and death. Researchers hope to simulate Parkinson's disease by breeding transgenic mice carrying these mutations. Among them, the composition and location of inclusion bodies in the A53T mutant mouse strain are similar to the aggregation of α-Syn in human neurons, and the pathological changes and behavioral abnormalities are progressive, which is more similar to the pathogenesis of human Parkinson's disease. Therefore, A53T transgenic mice are widely used as Parkinson's disease model mice. [6] However, there is currently no Parkinson's disease mouse model that has both Parkinson's disease pathological characteristics and cognitive impairment symptoms, so it is necessary to study a model that can more comprehensively simulate Parkinson's disease.

[0004] References

[0005] [1]Baiano C,Barone P,Trojano L,et al.Prevalence and clinical aspectsof mild cognitive impairment in Parkinson's disease:A meta-analysis.MovDisord.2020;35(1):45-54.

[0006] [2]Stoker TB,Greenland JC.Parkinson’s Disease:Pathogenesis andClinical Aspects.Brisbane(AU):Codon Publications;2018Dec 21.

[0007] [3]Spillantini MG,Schmidt ML,Lee VM,et al.Alpha-synuclein in Lewybodies.Nature.1997;388(6645):839-40

[0008] [4]Ferese R,Modugno N,Campopiano R,et al.Four Copies of SNCAResponsible for Autosomal Dominant Parkinson's Disease in Two ItalianSiblings.Parkinsons Dis.2015;2015:546462.

[0009] [5]Seo SH,Bacolla A,Yoo D,et al.Replication-Based Rearrangements Area Common Mechanism for SNCA Duplication in Parkinson's Disease.MovDisord.2020;35(5):868-876.

[0010] [6]Giasson BI, Duda JE, Quinn SM, Zhang B, Trojanowski JQ, Lee VM. Neuronalalpha-synucleinopathy with severe movement disorder in mice expressing A53Thuman alpha-synuclein. Neuron. 2002; 34(4):521-33. Summary of the invention

[0011] The primary purpose of the present invention is to provide a method for constructing a mouse model of Parkinson's disease cognitive impairment.

[0012] Another object of the present invention is to provide an application of the method for constructing the above-mentioned Parkinson's disease cognitive impairment mouse model.

[0013] The purpose of the present invention is achieved through the following technical solutions:

[0014] A method for constructing a Parkinson's disease cognitive impairment mouse model comprises the following steps: based on A53T α-Syn mice, the TREM2 gene is further inactivated to obtain a Parkinson's disease cognitive impairment mouse model with pathological deposition of hippocampal α-Syn and obvious cognitive impairment characteristics.

[0015] The inactivation is achieved by knocking out the TREM2 gene through a gene editing method; preferably, exon 2 and exon 3 fragments of the TREM2 gene are knocked out.

[0016] The inactivation of the TREM2 gene on the basis of A53Tα-Syn mice is carried out by the following steps: knocking out the TREM2 gene of wild-type mice by gene editing, mating the mice successfully knocked out with wild-type mice, and obtaining TREM2 + / - heterozygous; TREM2 + / - Heterozygotes were mated with A53Tα-Syn mice to select TREM2 + / - Multiple rounds of mating of α-Syn / A53T mice resulted in the establishment of a mouse model of cognitive impairment in Parkinson's disease, TREM2, which has pathological deposition of α-Syn in the hippocampus and obvious features of cognitive impairment. - / - A53Tα-Syn.

[0017] The gene fragment targeted by gene editing is preferably exon 2 and 3 of the TREM2 gene.

[0018] The sgRNA sequence in the gene editing is preferably as follows:

[0019] TREM2-L4: 5'-CAAGAACAGAGACACGAGAGG-3';

[0020] TREM2-R5: 5'-ATGTGGAAGAGCTCATCGGGG-3'.

[0021] The specific operation steps of the gene editing are preferably as follows: sgRNA and capped Cas9 mRNA are injected into wild-type mouse fertilized eggs by microinjection, and then transferred into pseudo-pregnant mother mice, and then the mice are screened after birth to obtain mice with successful knockout.

[0022] The concentration of the sgRNA is preferably 5 to 30 ng / μL; more preferably 15 ng / μL.

[0023] The concentration of the capped Cas9 mRNA is preferably 50-100 ng / μL; more preferably 75 ng / μL.

[0024] The sgRNA and the Cas9 mRNA are preferably mixed at a concentration ratio of 1:5 to 10; more preferably at a concentration ratio of 1:5.

[0025] The wild-type mice are preferably C57BL / 6 mice; more preferably C57BL / 6J mice.

[0026] The adult A53Tα-Syn mice are human mutant A53Tα-Syn Parkinson's disease transgenic mice.

[0027] The above-mentioned method for constructing a mouse model of Parkinson's disease cognitive impairment plays an important role in studying the mechanism and / or therapeutic targets of Parkinson's disease cognitive impairment.

[0028] Beneficial effects of the present invention:

[0029] The present invention uses gene editing technology to knock out the TREM2 gene of wild-type mice, and obtains a mouse model of Parkinson's disease cognitive impairment (genotype TREM2) by mating and screening with A53Tα-Syn mice. - / - / A53Tα-Syn). The invention verifies the occurrence of cognitive impairment in the model mice through multiple indicators of behavioral experiments and molecular biology experiments, has strong repeatability and genetic stability, provides a reliable method for constructing a Parkinson's disease cognitive impairment model, provides an important basis for exploring the mechanism of action and treatment strategy of Parkinson's disease cognitive impairment, and lays a foundation for clinical relief of Parkinson's disease cognitive impairment symptoms and drug development. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 TREM2 in Example 1 - / -Flowchart for modeling of / A53T α-Syn mice.

[0031] Figure 2 Schematic diagram of knocking out the TREM2 gene in Example 1.

[0032] Figure 3 It is a diagram of the TREM2-A53T mouse genotype identification results in Example 1; wherein A is a diagram of the TREM2 gene knockout identification results, B is a diagram of the TREM2 wild gene identification results, C is a diagram of the A53T gene identification results, and D is a summary diagram of the identification results.

[0033] Figure 4 For C57BL / 6 and TREM2 in Example 2 - / - 、A53Tα-Syn、TREM2 - / - / A53Tα-Syn mice novel object recognition experiment results; A is a schematic diagram of the novel object recognition experiment, and B is a graph showing the cognitive index results of the novel object recognition experiment for each group of mice.

[0034] Figure 5 For C57BL / 6 and TREM2 in Example 2 - / - 、A53Tα-Syn、TREM2 - / - / A53Tα-Syn mice Morris water maze test results; wherein, A is the percentage (%) of the time each group of mice stayed in the target quadrant during the exploration period, and B is the latency (s) of each group of mice to find the platform during the positioning cruise period.

[0035] Figure 6 For C57BL / 6 and TREM2 in Example 2 - / - 、A53Tα-Syn、TREM2 - / - Typical movement trajectory of / A53Tα-Syn mice during the Morris water maze spatial exploration phase.

[0036] Figure 7 For C57BL / 6 and TREM2 in Example 3 - / - 、A53Tα-Syn、TREM2 - / - Detection diagram of α-Syn level in the cerebral cortex of / A53Tα-Syn mice; A and C are the expression levels of oligomeric α-Syn, phosphorylated α-Syn and monomeric α-Syn in the left and right cerebral cortices of four groups of mice, respectively; B and D are the quantitative results of monomeric α-Syn, oligomeric α-Syn and phosphorylated α-Syn in groups A and C; β-Tubulin is the internal reference, Oligomer(α-Syn) represents oligomeric α-Syn, p-α-Syn represents phosphorylated α-Syn, and Monomer(α-Syn) represents monomeric α-Syn.

[0037] Figure 8 For C57BL / 6 and TREM2 in Example 3 - / - 、A53Tα-Syn、TREM2 - / - Detection diagram of α-Syn level in hippocampal tissue of / A53T α-Syn mice; A and C are the expression level diagrams of oligomeric α-Syn, phosphorylated α-Syn and monomeric α-Syn in the left and right hippocampal tissues of four groups of mice, respectively; B and D are the quantitative results of monomeric α-Syn, oligomeric α-Syn and phosphorylated α-Syn in groups A and C; β-Tubulin is the internal reference, Oligomer (α-Syn) represents oligomeric α-Syn, p-α-Syn represents phosphorylated α-Syn, and Monomer (α-Syn) represents monomeric α-Syn.

[0038] Fig. 9 For C57BL / 6 and TREM2 in Example 3 - / - 、A53Tα-Syn、TREM2 - / - Detection diagram of α-Syn level in the striatum of / A53Tα-Syn mice; A and C are the expression level diagrams of oligomeric α-Syn, phosphorylated α-Syn and monomeric α-Syn in the left and right striatum of four groups of mice, respectively; B and D are the quantitative results of monomeric α-Syn, oligomeric α-Syn and phosphorylated α-Syn in groups A and C; β-Tubulin is the internal reference, Oligomer(α-Syn) represents oligomeric α-Syn, p-α-Syn represents phosphorylated α-Syn, and Monomer(α-Syn) represents monomeric α-Syn.

[0039] Fig.10 For C57BL / 6 and TREM2 in Example 4 - / - 、A53Tα-Syn、TREM2 - / - Figure 3: Immunofluorescence detection of TH expression in the substantia nigra of / A53Tα-Syn mice; A is a fluorescence image of TH expression levels in the substantia nigra of four groups of mice, and B is a quantitative result of TH expression in each group in A.

[0040] Fig.11 For C57BL / 6 and TREM2 in Example 4 - / - 、A53Tα-Syn、TREM2 - / - Figure 3 Immunohistochemical detection of TH expression in the substantia nigra of / A53Tα-Syn mice; A is the histochemical image of TH expression levels in the substantia nigra of four groups of mice, and B is the quantitative result of TH expression in each group in A.

[0041] Fig.12 For C57BL / 6 and TREM2 in Example 5 - / -、A53Tα-Syn、TREM2 - / - The results of behavioral tests related to movement disorders in Parkinson's disease in / A53Tα-Syn mice; A is the result of the duration of the wire suspension experiment, and B is the result of the duration of the rotarod experiment in four groups of mice. DETAILED DESCRIPTION

[0042] To make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be described clearly and completely below. It should be noted that the equipment and raw materials used can be purchased from the market or are commonly used in the art. The methods in the following embodiments, unless otherwise specified, are conventional methods in the art.

[0043] Some of the materials used are listed below:

[0044]

[0045]

[0046] The composition of the lysis buffer is as follows: RIPA, PMSF and phosphatase inhibitor are mixed in a volume ratio of 100:1:2, wherein RIPA is RIPA lysis buffer from Beyotime, catalog number P0013B; PMSF is purchased from Beyotime, catalog number: ST506-2; phosphatase inhibitor is phosphatase inhibitor mixture A50X from Beyotime, catalog number: P1082.

[0047] Example 1

[0048] The construction process of the mouse model of Parkinson's disease cognitive impairment is as follows Figure 1 As shown in Figure 2, the process of gene editing the TREM2 gene is as follows Figure 2 As shown:

[0049] (1) Multiple sgRNA target sites were designed in the introns near the ends of exon 2 (exon2) and exon 3 (exon3) of the TREM2 gene (its sequence is shown in NCBI's Gene ID: 54209), and the in vitro enzymatic activity of the above sgRNA target sites was detected using the Beijing Weishanglide Cas9 in vitro enzymatic cleavage method gRNA target efficiency detection kit (Cat. No.: VK007).

[0050] (2) Select highly active sgRNA (TREM2-L4 and TREM2-R5 mixed in a molar ratio of 1:1) and dilute it with capped Cas9 mRNA (Beijing Weishanglide Biotechnology Co., Ltd., catalog number: R001L) to make the final concentrations of sgRNA and Cas9 mRNA 15 ng / μL and 75 ng / μL, respectively. Pipette 3 μL into the injection needle and inject it into the in vitro fertilized eggs of wild-type C57BL / 6 mice (source: Beijing Weishanglide Biotechnology Co., Ltd., 8 weeks old) via cytoplasm. The local area of ​​the fertilized egg injected shows obvious swelling, but the fertilized egg itself does not show obvious swelling.

[0051] TREM2-L4: 5'-CAAGAACAGAGACACGAGAGG-3';

[0052] TREM2-R5: 5'-ATGTGGAAGAGCTCATCGGGG-3'.

[0053] (3) In vitro fertilized eggs (IVF) were transplanted into pseudopregnant mice and were born 3 weeks later. At 2 weeks of age, genomic DNA was extracted from the mouse tails. Primers M-KO-T13-FP and M-KO-T13-RP (sequences shown in Table 1) were used to detect the sequence of the pre-mutation (i.e., the sequence of exon2 and exon3 to be knocked out) by PCR. The PCR reaction system was as follows: KOD FX (TOYOBO, KFX-201) 0.5 μL, dNTPs at a concentration of 2.5 mmol / L 5 μL, 2× buffer 12.5 μL, genomic DNA 2 μL, upstream primer at a concentration of 10 μmol / L 1 μL, downstream primer at a concentration of 10 μmol / L 1 μL, double distilled water to make up to 25 μL; PCR reaction conditions were as follows: 94°C for 2 min; 98°C for 10 sec, 60°C for 30 sec, 68°C for 60 sec, 35 cycles; 68°C for 10 min; 16°C for 2 min. The mutant mice were confirmed to be mutated and then the specific sequence deleted was confirmed to be not a multiple of 3 by first-generation sequencing, thus confirming that the founder mice were obtained.

[0054] Table 1 Primer sequences and sizes of target PCR products

[0055]

[0056] (4) The obtained first-generation mice were mated with wild-type C57BL / 6 mice (Beijing Weishanglide Biotechnology Co., Ltd., 8 weeks old) to obtain stable genetic F1 TREM2 knockout mice TREM2 + / - .

[0057] (5) A53Tα-Syn Parkinson's disease transgenic mice (Beijing Weishanglide Biotechnology Co., Ltd., 8 weeks old) were cross-linked with TREM2 + / - Mice were mated with each other and bred; the resulting mice were then mated multiple times to obtain TREM2 + / - / A53T α-Syn and TREM2 - / - / A53Tα-Syn mice, Trem2-KO genotype identification, the specific identification method is as follows:

[0058] 1) Extraction of mouse DNA: Cut 0.3-0.5mm mouse tails around 7 days after birth and place in a 1.5mL EP tube containing 50μL lysis solution, centrifuge at 13300 rpm for 30s, place in a 60℃ oven overnight, then take out the sample and cool to room temperature, centrifuge at 13300 rpm for 10s to allow the liquid in the tube to sink to the bottom. Add 7μL of neutralizing solution to each tube of sample and centrifuge at 13300 rpm for 5min. Take the supernatant and test the DNA concentration.

[0059] The lysis solution was prepared as follows: 0.4 g NaOH was dissolved in 200 mL deionized water to obtain a NaOH solution; the aforementioned NaOH solution and a proteinase K solution having a concentration of 20 mg / mL were mixed in a volume ratio of 50:0.2 to obtain a lysis solution.

[0060] Preparation of neutralization solution: 18.17 g Tris-HCl was dissolved in 150 mL deionized water, and the final pH was 8.0.

[0061] 2) Agarose gel electrophoresis detection: The product obtained according to the PCR system and procedure described in step (3) is detected. The detection result is as follows: Figure 3 As shown: Figure 3 The appearance of the target band in A indicates that at least one chromosome of the tested mouse has had TREM2 gene exon2-3 knocked out successfully. Figure 3 If the target band does not appear in B, it means that there is no wild-type TREM2 gene on both chromosomes of the tested mouse. The combination of A and B indicates that the TREM2 genes on both chromosomes of the tested mouse are knocked out, that is, the mouse is homozygous TREM2(- / -). Figure 3 The appearance of the target band in C indicates that the mouse has the A53T mutation, i.e., A53T(+).

[0062] Example 2

[0063] Behavioral testing related to cognitive impairment in Parkinson's disease

[0064] Experimental animals: A total of 39 mice were used in the experiment, including 9 C57BL / 6 mice, 9 TREM2 - / - 10 mice, 9 A53Tα-Syn mice, TREM2- / - 11 / A53Tα-Syn mice (prepared in Example 1).

[0065] (1) New object recognition and detection:

[0066] Experimental method: First, prepare an opaque box (40cm×40cm×40cm, length×width×height). Before the experiment begins, make sure that the experimental box is clean and odorless. Each mouse needs to clean the box after the experiment. One hour before the experiment begins, place the mouse in the behavioral laboratory to familiarize it with the environment. Adaptation period: The mouse is quickly placed in the edge area of ​​the experimental box facing the box wall, and the mouse is allowed to move freely for 10 minutes; Familiarization period: Place two objects with exactly the same color and shape and no odor in the experimental box (A / B, make sure they are not pushed). The two identical objects (radius of 1.5cm) are 5.5cm away from the side wall. Place the mouse facing the box wall and its back facing the objects in the experimental box from the middle of the two objects. Allow it to explore freely for 5 minutes before taking out the mouse. Testing phase: Replace one of the two identical objects with an object of inconsistent shape and color and place it in the experimental box (AC / BC, make sure the object has no odor and is not pushed, such as Figure 4 As shown in A in the figure, the two different objects were 5.5 cm away from the side wall; the mouse was placed in the experimental box with its face facing the box wall and its back facing the object from the middle of the two objects, and the mouse's activities were recorded within 5 minutes. When the mouse faced the object and the distance between the nose and the object was less than 2 cm, it was defined as exploration, while the mouse standing on the object without moving was not considered exploration. The memory ability of mice was evaluated using the recognition index (RI).

[0067] RI = N / (N+F) × 100%;

[0068] Among them, N is the time / number of exploration of the new object, and F is the time / number of exploration of the old object.

[0069] Experimental results: C57BL / 6, TREM2 - / - 、A53Tα-Syn、TREM2 - / - The results of cognitive index of the novel object recognition experiment in the four groups of mice with A53Tα-Syn are as follows: Figure 4 As shown in B, at this stage TREM2 - / - Compared with C57BL / 6 and TREM2 / A53Tα-Syn mice, - / - For A53Tα-Syn mice, their preference for new objects was significantly reduced, indicating that their memory function was impaired.

[0070] 2. Morris water maze experiment:

[0071] Experimental methods:

[0072] Spatial exploration experimental method: Place the animal in the water with its head facing the pool wall, and randomly place it in one of the four starting positions of east, west, south, and north. Record the time (s) it takes for the animal to find the underwater platform. Each animal is trained 4 times a day, with an interval of 15 to 20 minutes between two training sessions, and the training is continued for 5 days. On the second day after the last acquisition training, the platform is removed and a 60-second exploration training is started. Place the animal in the water from the opposite side of the original platform quadrant. Record the animal's movement trajectory and the time spent in the target quadrant (the quadrant where the platform was originally placed) as a test indicator of spatial memory.

[0073] Method of positioning cruising experiment: Place the platform in the opposite quadrant of the original platform, and train four times a day in the same way as before. Record the time (latency) for the animal to find the platform each time, and use it as a working memory test indicator.

[0074] Experimental results: C57BL / 6, TREM2 - / - 、A53T、TREM2 - / - The results of Morris water maze test of four groups of A53T mice are as follows Figure 5 and Figure 6 Shown: TREM2 - / - / A53Tα-Syn mice spent significantly less time in the target quadrant during the spatial exploration period than C57BL / 6 and TREM2 - / - , A53T mice (such as Figure 5 ), where on the fifth day of the experiment, TREM2 - / - / A53T mice and A53T mice showed significant differences in TREM2 - / - The latency of / A53Tα-Syn mice to find the platform during the positioning cruise period was significantly higher than that of C57BL / 6 and TREM2 - / - , A53Tα-Syn mice (e.g. Figure 5 TREM2 - / - The movement trajectory of / A53Tα-Syn mice tended to be marginalized compared with other groups of mice, indicating that TREM2 - / - / A53Tα-Syn mice have poor spatial memory ( Figure 6 The above experimental results show that TREM2 - / - Compared with C57BL / 6 and TREM2 - / - , A53Tα-Syn mice were reduced.

[0075] Example 3

[0076] Molecular biological detection of cognitive impairment in Parkinson's disease

[0077] Experimental method: The levels of α-Syn oligomers, phosphorylated α-Syn and α-Syn monomer proteins were detected by protein immunoblotting. The specific steps were as follows: one week after the behavioral experiment, 3 mice were randomly selected from each group to collect samples of the left and right cerebral cortex, hippocampus and striatum, and 1% pentobarbital was prepared with normal saline, and then the mice were anesthetized by intraperitoneal injection of pentobarbital. After the anesthesia effect was satisfactory, the chest cavity was cut open, and the needle was inserted into the left ventricle until the ascending aorta and then fixed. The right atrial appendage was cut open, and the blood was quickly flushed clean by cardiac perfusion with pre-cooled normal saline. The left and right cerebral cortex, hippocampus and striatum were separated on ice for protein extraction, and placed in pre-cooled EP tubes respectively and stored at -80℃ for later use. The mouse tissue was mixed with the lysis buffer at a ratio of 1 mg: 10 μL, and lysed on ice for 30 minutes. Two magnetic beads were added to each EP tube, and ground twice in a grinder (50 Hz), each time for 5 minutes. After cooling in ice, ultrasonic crusher was used for 5 seconds / time, and repeated 3 times, each time for 10 seconds, with a frequency of 30%. The EP tube was then placed in a pre-cooled centrifuge, centrifuged at 4°C and 15000g for 10 minutes, and the supernatant was collected. The total protein was then separated by 12.5-15% SDS-PAGE polyacrylamide gel electrophoresis, and then transferred to a PVDF membrane. After blocking with 5% skim milk on a shaker for 2 hours, the PVDF membrane was transferred to the pre-prepared primary antibody working solution [abcam ab138501 as the primary antibody for α-Syn oligomers and α-Syn monomers (dilution ratio 1:1000), abcam ab51253 as the primary antibody for phosphorylated α-Syn (dilution ratio 1:1000); the diluent was TBST buffer (pH7.5, Tween20 0.1%, the same below)], and incubated overnight on a shaker at 4°C. After the primary antibody incubation, the membrane was washed with TBST buffer for 3 times, 10 minutes each time. After washing, the PVDF membrane was removed and placed in the secondary antibody working solution (Yamei LF102 as the secondary antibody, diluted by volume ratio 1:2000, the diluent was TBST buffer) and incubated on a shaker at room temperature for 1 hour, and the above washing steps were repeated. Finally, ECL color exposure (YEASEN36208ES60) was used to analyze the grayscale value of each band using Image J software to analyze the levels of oligomeric α-Syn, phosphorylated α-Syn, and monomeric α-Syn in the cortex, hippocampus, and striatum on both sides of the left and right sides. Figure 7 , Figure 8 and Fig. 9 shown.

[0078] Experimental results: Figure 7 A and C in Figure 8 A and C in Fig. 9 The color depth of the bands in A and C in Figure 1 represents the abundance of protein expression, and the darker the color, the higher the level of the protein; Figure 7 B and D in Figure 8 B and D in Fig. 9 B and D in the table are the corresponding statistical results. The pathological characteristics of Parkinson's disease are the progressive loss of dopaminergic neurons in the substantia nigra and the formation of Lewy bodies. α-Syn is the main component of Lewy bodies and is a key protein in Parkinson's disease. Pathological α-Syn will invade the central and peripheral nervous systems, accumulate in neurons, and cause a wide range of motor and non-motor clinical symptoms. The brain areas related to cognitive function mainly include the neocortex, hippocampus, striatum, etc. Therefore, the accumulation of α-Syn in the brain areas related to cognitive function will affect the function of the brain area and lead to cognitive impairment. Figure 7 , Figure 8 , Fig. 9 As shown, compared with the A53Tα-Syn group, TREM2 - / - The levels of monomeric α-Syn, oligomeric α-Syn, and phosphorylated α-Syn in the cortex, hippocampus, and striatum of / A53T α-Syn mice were significantly increased, indicating that TREM2 - / - / A53Tα-Syn mice had higher α-Syn content in cognitive brain regions and decreased cognitive function compared with A53Tα-Syn mice.

[0079] Example 4

[0080] Microscopic pathological examination of motor brain area in Parkinson's disease

[0081] Experimental method: The expression of TH (tyrosine hydroxylase) in the substantia nigra was detected by immunofluorescence and immunohistochemistry. The preliminary steps of the two were the same, specifically: one week after the end of the behavioral experiment, 3 mice were randomly selected from each group for cardiac perfusion, 1% pentobarbital was prepared with normal saline, and then the mice were anesthetized by intraperitoneal injection of pentobarbital. After the anesthesia effect was satisfactory, the chest cavity was cut open, the needle was inserted into the left ventricle until the ascending aorta was fixed, the right atrial appendage was cut open, and the blood was quickly flushed out by cardiac perfusion with pre-cooled normal saline, and then 4% paraformaldehyde was used to continue transcardial perfusion, and then the mouse brain was completely taken out and immersed in a test tube containing 4% paraformaldehyde and stored at -20°C. The fixed tissue was sent to the company for paraffin embedding and sectioning. The obtained paraffin sections were placed in a 60℃ oven for 30 minutes (observe whether the paraffin turns into oil droplets when the time is up. If not, the baking time can be extended to 1 hour); then the sections were immersed in dewaxing transparent liquid (G1128-1L) for 30 minutes, and dewaxing was repeated twice. Then, the sections were immersed in gradient ethanol (absolute ethanol - absolute ethanol - 95% - 85% - 75% ethanol) and distilled water for 5 minutes each for hydration. Prepare sodium citrate antigen repair solution [one pack of sodium citrate powder preparation (BL604A), double distilled water to 1000mL], place the slice in sodium citrate antigen repair solution and put it in a microwave oven for 10 minutes on medium heat, then adjust to high heat and boil for 5 minutes (high heat is 100%, medium heat is 60-70%, pay attention to observe whether the tissue on the slice is immersed during the repair process, and increase the volume of sodium citrate antigen repair solution appropriately), take it out, and cool it naturally to room temperature. Circle the tissue to be tested with a histochemical pen, add 1-2 drops of 0.1% Triton X-100 to break the membrane for 15 minutes, and wash with PBS for 5 minutes×3.

[0082] (1) Immunofluorescence

[0083] Endogenous peroxidase was blocked using the rabbit / mouse universal Streptavidin-HRP kit (CW2069S), 1-2 drops of Solution A blocking solution were added, incubated at room temperature for 10 minutes, and washed with PBS for 5 minutes × 3; 1-2 drops of Solution B, i.e. normal sheep serum working solution for blocking, were added, incubated at room temperature for 10 minutes, and dried. 1-2 drops of the pre-prepared primary antibody working solution (ab137869 dilution ratio 1:250, diluent is BSA catalog number 37525, the same below), the slices were placed in a humidified box and incubated overnight at 4°C.

[0084] The following are all light-proof operations:

[0085] The next day, wash with PBS for 5 min × 3 times, add 1-2 drops of secondary antibody working solution (A11037 dilution ratio 1:500 dilution is BSA), incubate at room temperature for 1 hour, wash with PBS for 5 min × 3 times, add 1-2 drops of DAPI (G1012-10ML) and incubate in the dark for 5 min, stain the nucleus of the specimen, soak in PBS and shake on a shaker for 5 min, wash away excess DAPI, absorb water with filter paper, and seal with anti-fluorescence quenching sealing agent (G1401-5ML) and store in a dry box. Observe and shoot with an inverted laser confocal microscope, and quantify and analyze the fluorescence intensity with Image J software later. The results are shown in Fig.10 shown.

[0086] Experimental results: Fig.10 The fluorescence intensity of A in the figure indicates the expression of related indicators. Red represents TH, and blue represents the cell nucleus. The brighter the color, the higher the expression level of the indicator. Fig.10 B in the figure is the corresponding statistical result. The pathological characteristics of Parkinson's disease are the progressive loss of dopaminergic neurons in the substantia nigra and the formation of Lewy bodies. The reduction of dopamine synthesis is the main cause of movement disorders in Parkinson's disease. TH is a key enzyme in dopamine synthesis. Its reduced expression in the substantia nigra can lead to movement disorders. Fig.10 As shown, C57BL / 6 vs. TREM2 - / - group, A53Tα-Syn vs. TREM2 - / - In the / A53Tα-Syn group, there was no significant difference in the expression of TH in the substantia nigra of mice, indicating that TREM2 knockout has no additional effect on the motor function of the model mice.

[0087] (2) Immunohistochemistry

[0088] Endogenous peroxidase blocking and antibody incubation were performed using the rabbit / mouse universal Streptavidin-HRP kit (CW2069S), 1-2 drops of Solution A blocking solution were added and incubated at room temperature for 10 minutes, and PBS was used for washing for 5 minutes × 3; 1-2 drops of Solution B, i.e. normal sheep serum working solution for blocking, were added and incubated at room temperature for 10 minutes, and then dried. 1-2 drops of the pre-prepared primary antibody working solution (ab137869 dilution ratio 1:500 dilution solution is BSA) were added, and the sections were placed in a humidified box and incubated overnight at 4°C.

[0089] The next day, wash with PBS for 5 min × 3 times, add 1-2 drops of Solution C, i.e., biotin-labeled goat anti-rabbit / mouse secondary antibody working solution, and incubate at room temperature for 10 minutes; wash with PBS for 5 min × 3 times, add an appropriate amount of Solution D, i.e., HRP-labeled streptavidin, and incubate at room temperature for 10 minutes; wash with PBS for 5 min × 3 times, mix DAB-A and DAB-B in the kit at a volume ratio of 1:19 to prepare DAB color development working solution, add an appropriate amount of DAB color development working solution (just enough to cover the tissue section to be colored) for color development (pay attention to avoid light, rinse with tap water to stop color development when brown is visible to the naked eye); after color development, stain with hematoxylin staining solution for 3 minutes, rinse with tap water; hydrochloric acid ethanol differentiation solution (75% EtOH + 0.1% 37% HCl) rinse, rinse with water (tap water is fine), and then rinse twice with double distilled water; prepare a slide rack, place the slides neatly, and place the slide rack in a 60℃ oven to bake for 5-10 minutes; drop an appropriate amount (10-20μL) of neutral resin on the slide, and cover the slide with a cover glass. After sealing, take pictures under a microscope. Image J software was used to quantify the expression of TH in the substantia nigra of each group. The results are shown in the figure. Fig.11 shown.

[0090] Experimental results: Fig.11 The depth of A staining in the figure represents the expression of TH, and the darker the color, the higher the TH expression; Fig.11 B in the figure is the corresponding statistical result. There was no significant difference in TH expression in the substantia nigra between the C57BL / 6 and TREM2- / - groups, and between the A53Tα-Syn and TREM2- / - / A53Tα-Syn groups, indicating that TREM2 knockout had no significant effect on the motor function of the model mice.

[0091] Example 5

[0092] Behavioral testing for movement disorders in Parkinson's disease

[0093] Experimental animals: A total of 39 mice participated in the experiment, including 9 C57BL / 6 mice, 10 TREM2- / - mice, 9 A53Tα-Syn mice, and 11 TREM2- / - / A53Tα-Syn mice (prepared in Example 1).

[0094] Wire suspension experiment:

[0095] Experimental method: The time it takes for the mouse to fall due to exhaustion is recorded. Use a wire cage lid and tape around the edges to prevent the mouse from walking off the edge. Place the animal on top of the cage lid and gently shake the lid to force the mouse to grab the wire. Then, slowly flip the lid over, high enough to prevent the mouse from jumping off, but not high enough to cause injury if it falls, and record the time it takes for the mouse to fall due to exhaustion.

[0096] Experimental results: The duration of the wire suspension experiment in four groups of mice: C57BL / 6, TREM2- / -, A53Tα-Syn, and TREM2- / - / A53Tα-Syn. Fig.12 As shown in A, at this stage, there was no significant difference in the time before falling for TREM2- / - / A53Tα-Syn mice compared with C57BL / 6, TREM2- / -, and A53Tα-Syn mice, indicating that their motor function was not decreased compared with the other three groups of mice.

[0097] Rotarod test:

[0098] Experimental method: The experimental adaptation stage is divided into three stages, with no time gap between the three stages: in the T1 stage, the rotating rod is accelerated from rest to a speed of 5rpm at an acceleration of 5rpm / min; in the T2 stage, the acceleration is changed to 10rpm / min, and it is accelerated to 15rpm and then rotated at a constant speed for 2min; in the T3 stage, it is continued to accelerate from 10rpm / min to 20rpm and then continue to rotate at a constant speed for 2min. If the mouse falls during the test, the falling time is recorded, and the experiment is restarted for the fallen mouse after 20min, and the time the mouse lasts on the rotating rod is recorded. During the adaptation experiment, the mouse is trained to strengthen its physical strength and coordination ability. In the formal experimental stage, the acceleration of each stage T1, T2, and T3 remains unchanged, and the uniform speed reaches 5r / min, 15r / min, and 18r / min at the end, respectively, and the duration of the mouse on the rotating rod is recorded.

[0099] Experimental results: The results of the rotarod duration test in four groups of mice: C57BL / 6, TREM2- / -, A53Tα-Syn, and TREM2- / - / A53Tα-Syn are as follows: Fig.12 As shown in B, there is no significant difference in the rotarod duration of TREM2- / - / A53Tα-Syn mice compared with TREM2- / - and A53Tα-Syn mice, indicating that the motor function of TREM2- / - / A53Tα-Syn mice is not decreased compared with TREM2- / - and A53Tα-Syn mice.

[0100] The statistical methods involved in the above experiments are as follows:

[0101] All statistical analyses were performed in IBM SPSS 20.0. If the measurement data conformed to the normal distribution after the Kolmogorov-Smirnov test, the two independent sample t test was used to compare the means between two samples; one-way analysis of variance was used to compare the means between multiple samples, and the Levene method was further used to test whether the variance was homogeneous. If the variance was homogeneous, the LSD method was used for multiple comparisons, otherwise the Dunnet's T3 method was used for multiple comparisons; if it did not conform to the normal distribution, the Kruskal-Wallis H or Mann–Whitney U test method was used; two-way ANOVA was used for different groups at different time points in the behavioral Morris water maze data. The count data were tested using the χ2 test.

[0102] It should be noted that the above content only illustrates the technical idea of ​​the present invention and cannot be used to limit the protection scope of the present invention. For ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications all fall within the protection scope of the claims of the present invention.

Claims

1. A method for constructing a mouse model of Parkinson's disease cognitive impairment, characterized in that The following steps are involved: Based on the A53T α-Syn mouse, the TREM2 gene was inactivated to obtain a Parkinson's disease cognitive impairment mouse model with pathological deposition of hippocampal α-Syn and obvious characteristics of cognitive impairment.

2. The method for constructing a mouse model of Parkinson's disease cognitive impairment according to claim 1, characterized in that: The inactivation is achieved by knocking out the TREM2 gene through gene editing methods.

3. The method for constructing a mouse model of Parkinson's disease cognitive impairment according to claim 1, characterized in that: The inactivation of the TREM2 gene on the basis of A53Tα-Syn mice is carried out by the following steps: knocking out the TREM2 gene of wild-type mice by gene editing, mating the mice successfully knocked out with wild-type mice to obtain TREM2+ / - heterozygotes; then mating the TREM2+ / - heterozygotes with A53Tα-Syn mice to select TREM2 + / - The TREM2 mouse model of Parkinson's disease with typical pathological deposition of α-Syn in the hippocampus and obvious features of cognitive impairment was obtained by multiple rounds of mating of A53Tα-Syn mice. - / - A53Tα-Syn.

4. The method for constructing a mouse model of Parkinson's disease cognitive impairment according to claim 3, characterized in that: The wild-type mice are C57BL / 6 mice; The gene fragments targeted by gene editing are exons 2 and 3 of the TREM2 gene.

5. The method for constructing a mouse model of Parkinson's disease cognitive impairment according to claim 3, characterized in that: The sgRNA sequence in the gene editing is as follows: TREM2-L4: 5'-CAAGAACAGAGACACGAGAGG-3'; TREM2-R5: 5'-ATGTGGAAGAGCTCATCGGGG-3'.

6. The method for constructing a mouse model of Parkinson's disease cognitive impairment according to claim 3, characterized in that: The specific operation steps of the gene editing are as follows: sgRNA and Cas9 mRNA are injected into mouse fertilized eggs by microinjection, and then transferred into pseudo-pregnant mother mice, and then the mice are screened after birth to obtain mice with successful knockout.

7. The method for constructing a mouse model of Parkinson's disease cognitive impairment according to claim 6, characterized in that: The concentration of the sgRNA is 5 to 30 ng / μL; The concentration of the Cas9 mRNA is 50-100 ng / μL; The sgRNA and the Cas9 mRNA are mixed at a concentration ratio of 1:5-10.

8. The method for constructing a mouse model of Parkinson's disease cognitive impairment according to claim 7, characterized in that: The concentration of the sgRNA is 15 ng / μL; The concentration of the Cas9 mRNA is 75 ng / μL; The sgRNA and the Cas9 mRNA are mixed at a concentration ratio of 1:

5.

9. Use of the method for constructing a mouse model of cognitive impairment in Parkinson's disease according to any one of claims 1 to 8 in studying the mechanism of action and / or therapeutic targets of cognitive impairment in Parkinson's disease.

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