Construction method and application of Lewy small body dementia animal model

Abstract

The invention discloses a construction method and application of a Lewy small body dementia animal model, and belongs to the technical field of animal model construction. The construction method comprises the following steps: obtaining a phosphatase and tensin homologous gene-induced kinase 1 gene knockout experimental animal A by using a gene editing technology, and carrying out hybrid breeding on the experimental animal A and an Alzheimer disease model experimental animal B simulating beta-amyloid protein (Abeta) pathology, and screening filial generations containing the Alzheimer disease related mutation and pink1 gene knockout at the same time. According to the constructed model, beta amyloid protein is deposited on the basis of pink1 gene knockout, beta-amyloid plaque (A beta) is used for inducing alpha-synuclein protein aggregation, Lewy bodies are widely generated in cortex and hippocampus, the occurrence and development process of DLB is simulated, and a reliable animal model is provided for disease mechanism research and treatment drug screening and development.

Description

Technical Field

[0001] The present invention relates to the technical field of animal model construction, and in particular to a method for constructing and using a Lewy body dementia animal model. Background Art

[0002] Dementia of Lewy bodies (DLB) is a neurodegenerative disease, the second largest type of degenerative dementia after Alzheimer's disease (AD), accounting for about 10-15% of the incidence of dementia. The main clinical manifestations include Parkinson's disease (PD)-like symptoms such as cognitive impairment, hallucinations, rapid eye movement sleep disorders, slow movements, etc., which may be accompanied by digestive system symptoms such as constipation and cardiovascular system symptoms such as arrhythmias. Imaging examinations using positron emission tomography (PET) technology can reveal reduced dopamine transport in the basal ganglia. The characteristic pathological changes are the formation of Lewy bodies in the brain, which are mainly composed of α-synuclein, and Lewy bodies are mainly distributed in the cortex of the whole brain.

[0003] Clinically, DLB has a high incidence rate. Due to the diverse clinical manifestations of onset, diagnosis is very difficult. In the early stages, it is often difficult to differentiate between Parkinson's disease, sleep disorders, digestive system abnormalities, schizophrenia, Alzheimer's disease and many other diseases. In addition, due to the possibility of arrhythmia, it is easy to cause cardiogenic shock and even sudden death. The pathogenesis of DLB ​​is mainly related to the formation of Lewy bodies by α-synuclein protein, but the formation, development and maturation mechanism of Lewy bodies is still unclear, resulting in the unknown occurrence and development mechanism of DLB. There is a lack of therapeutic drugs for DLB. Currently, only symptomatic treatment can be performed clinically, such as the use of acetylcholinesterase inhibitors, a therapeutic drug for Alzheimer's disease, to treat dementia symptoms.

[0004] Therefore, there is an urgent need in this field to study the pathogenesis of DLB, the diagnostic methods, and the preventive and therapeutic drugs. The key method is to construct a DLB animal disease model to study the pathogenesis of DLB, find clinically applicable diagnostic biomarkers, and screen therapeutic drugs.

[0005] There is currently no animal model for DLB. In existing studies that require the use of the DLB model, the animal model of Parkinson's disease was "borrowed" mainly because Parkinson's disease also has Lewy body formation caused by α-synuclein protein aggregation. Both DLB and PD are Lewy body diseases, but the distribution of Lewy bodies in the two diseases is different. The Lewy bodies of DLB ​​are widely distributed, mainly occurring in the cerebral cortex and limbic system, with a small number found in the brainstem. α-synuclein protein aggregation can also be found in nerve fibers of peripheral organs such as the heart, intestines, and skin, while Lewy bodies of PD only occur in dopaminergic neurons in the substantia nigra (Guidelines for the diagnosis and treatment of dementia with Lewy bodies in China [J]. Chinese Journal of Geriatrics, 2021, 40(12):12.; 2018 Guidelines for the diagnosis and treatment of dementia and cognitive impairment in China (I): Dementia and its classification and diagnostic criteria [J]. Chinese Medical Journal, 2018, 98(13):6.).

[0006] At present, the models used for DLB research are mainly transgenic mouse models and cell models. Among them, the mouse model usually requires mice with overexpression of α-synuclein protein, combined with specific protein knockout or overexpression, and finally requires injection of exogenous fibrotic α-synuclein protein into the brain as a "seed". Fibrotic α-synuclein protein (α-Syn PFFs) can induce the aggregation of α-synuclein protein in mouse neurons and produce Lewy bodies (Visanji NP, et al. α-Synuclein-Based Animal Models of Parkinson's Disease: Challenges and Opportunities in a New Era. Trends Neurosci. 2016 Nov; 39 (11): 750-762.). The cell model of DLB ​​mainly uses exogenous fibrotic α-synuclein protein as a "seed", adds primary cultured rat or mouse neurons, and observes the aggregation of α-synuclein protein in inclusion bodies in neurons. This cell model is also used in PD research.

[0007] The advantages of the above models are: ① There are different degrees of neurodegeneration; ② There are some clinical and biochemical characteristics of PD, DLB, and MSA, such as α-Syn oligomers and fibers; ③ Detect the transport of extracellularly injected α-Syn along the neuroanatomical pathway; Disadvantages are: ① No dopaminergic neuron damage was detected; ② The α-Syn mutant model is not applicable to DLB with misfolded α-Syn; ③ α-Syn PFFs are required for accelerated induction, which is not required for clinical DLB; ④ The clinical phenotype is incomplete and there is no key phenotype of dementia; ⑤ It is impossible to simulate the spontaneous and widespread distribution of clinical Lewy bodies.

[0008] An effective animal model is an important tool for studying disease mechanisms and therapeutic drugs. An ideal DLB model should not overexpress α-synuclein protein, and the gene interference should be detectable in clinical patients. The model animal should be able to produce a dementia phenotype and partially or completely simulate the clinical manifestations of DLB, such as irritability, constipation, sudden death, etc. The most important thing is that pathological examination can find widely distributed Lewy bodies in the cortex and hippocampus. However, to date, there is no animal model that can simulate the occurrence and development of DLB, and it is difficult to conduct in-depth research on DLB. Therefore, it is very necessary to establish an animal model of DLB ​​with an early phenotype and a development process. Summary of the invention

[0009] The purpose of the present invention is to construct an experimental animal model that can simulate the occurrence and development process of Lewy body dementia, and to provide a reliable animal model for disease mechanism research and therapeutic drug screening and development.

[0010] To achieve the above object, the present invention adopts the following technical solution:

[0011] The present invention provides a method for constructing an animal model of Lewy body dementia, comprising the following steps:

[0012] (1) Gene editing technology was used to delete the expression of the phosphatase and tensin homologous gene-induced kinase 1 gene pink1 in the genome of experimental animals to obtain pink1 gene knockout homozygous (pink1 - / - ) or heterozygous (pink1 + / - ) Experimental animal A;

[0013] (2) Crossbreeding the pink1 gene knockout homozygous or heterozygous experimental animal A with an Alzheimer's disease model experimental animal B, wherein the Alzheimer's disease model experimental animal has an Alzheimer's disease-associated mutation in its genome and exhibits β-amyloid plaque pathology, to obtain offspring, from which offspring containing both the Alzheimer's disease-associated mutation and the pink1 gene knockout heterozygous or homozygous are screened, thereby obtaining the Lewy body dementia animal model.

[0014] Studies have shown that phosphatase and tensin homologue induced putative kinase 1 (PINK1) gene knockout animals exhibit significant motor defects, age-dependent loss of substantia nigra dopaminergic neurons, mitochondrial dysfunction, loss of locus coeruleus neurons, and formation of α-Syn aggregates in different brain regions, which are consistent with the manifestations of clinical Parkinson's disease (PD) patients.

[0015] The present invention crossbreeds pink1 gene knockout animals with Alzheimer's disease (AD) animals simulating beta-amyloid protein (Aβ) pathology, and finds that offspring animals with pink1 gene knockout and Alzheimer's disease-related mutations exhibit typical clinical symptoms of Lewy body dementia.

[0016] Taking the mouse DLB model as an example, the clinically relevant phenotypes it exhibits include: (1) early sudden death; (2) constipation and difficulty defecation; (3) dilated cardiomyopathy; (4) dementia; (5) neurological damage; (6) extensive Lewy bodies in the brain; and (7) Lewy bodies in the intestines and heart.

[0017] The offspring can be the first generation obtained by hybridization of the parents; or the nth generation obtained by hybridization of the offspring and the offspring or the offspring and the parents, where n≥2. For example, the parents hybridize to obtain the F1 generation, and the offspring individuals containing both Alzheimer's disease-related mutations and pink1 gene knockout heterozygotes are screened in the F1 generation to obtain the Lewy body dementia animal model. Alternatively, the offspring individuals in the F1 generation that simultaneously contain an Alzheimer's disease-associated mutation and a pink1 gene knockout heterozygote may be bred with the offspring individuals that only contain a pink1 gene knockout heterozygote for F2 generation breeding; or the offspring individuals in the F1 generation that simultaneously contain an Alzheimer's disease-associated mutation and a pink1 gene knockout heterozygote may be bred with the parent pink1 gene knockout homozygote or pink1 gene knockout heterozygote for F2 generation breeding; or two offspring individuals in the F1 generation that simultaneously contain an Alzheimer's disease-associated mutation and a pink1 gene knockout heterozygote may be bred with the F2 generation, and then the offspring individuals that simultaneously contain an Alzheimer's disease-associated mutation and a pink1 gene knockout heterozygote or homozygote may be screened in the F2 generation, to obtain the Lewy body dementia animal model. The F2 generation may then be bred in the above manner.

[0018] Furthermore, the experimental animals used to construct the animal model of Lewy body dementia in the present invention can be experimental animals commonly used in research in the field, and experimental animals A and experimental animals B are animals of the same species, including but not limited to one of mice, rats, guinea pigs, and macaques.

[0019] In the construction method provided by the present invention, gene editing technology is used to knock out the pink1 gene, so that the expression of the pink1 gene in the genome of the experimental animal is lost.

[0020] The gene editing technology can adopt gene knockdown technology known in the art, including but not limited to: gene targeting technology using embryonic stem cells, CRISPR / Cas9 technology, zinc finger nuclease technology, transcription activator-like effector nuclease technology or homing endonuclease.

[0021] Specifically, the Gene ID of the mouse pink1 gene is 68943, and the CDS sequence is shown in SEQ ID NO.1; the Gene ID of the rat pink1 gene is 298575, and the CDS sequence is shown in SEQ ID NO.2; the Gene ID of the guinea pig pink1 gene is 100720253, and the CDS sequence is shown in SEQ ID NO.3; the Gene ID of the macaque pink1 gene is 706037, and the CDS sequence is shown in SEQ ID NO.4.

[0022] Preferably, the pink1 gene in the animal genome is knocked out using CRISPR-Cas9 technology, and a pair of sgRNA sequences are designed to target two segments of exon 6 of the pink1 gene, respectively. Removal of exon 6 can reduce pink1 mRNA transcription and cause the expressed PINK1 to lose its kinase activity.

[0023] Preferably, for mouse pink1 gene knockout, the nucleotide sequence of the sgRNA sequence is 5'-GACAGCCATCTGCAGAGAGG-3' and 5'-GCAGGCAGGACTCACCTC AG-3'.

[0024] In the present invention, the CRISPR-Cas9 technology targeting sites are not limited to the above-mentioned sites, and any targeting sites that can reduce pink1 gene expression or inactivate enzyme activity are applicable to the present invention, such as exons 2 and 3 of the pink1 gene.

[0025] The Alzheimer's disease model experimental animal used in the construction method provided by the present invention refers to an animal model obtained by genetic engineering technology with mutations associated with Alzheimer's disease and showing β-amyloid plaque pathology. The experimental animal can use a commercial AD animal model, or it can be constructed with reference to literature reports.

[0026] The mutations associated with Alzheimer's disease include but are not limited to mutations occurring in the amyloid precursor protein (APP) gene and / or presenilin 1 gene sequence. The present invention can also use experimental animal models in which other genes are mutated and produce Aβ pathological phenotypes.

[0027] Preferably, the experimental animal B is a transgenic animal that overexpresses a mutant human amyloid precursor protein and / or a mutant human presenilin 1 in animal neurons. The transgenic animal overexpresses mutant human amyloid precursor protein and human presenilin 1 in neurons, resulting in an increase in the longer form of amyloid β (the main component of amyloid deposits found in the brains of AD patients).

[0028] The human amyloid precursor protein has three common subtypes: APP770, APP695 and APP751, which are generated by alternative splicing of exons 7 and 8, and their amino acid sequences are shown in SEQ ID NO. 5, SEQ ID NO. 6 and SEQ ID NO. 7, respectively. The human amyloid precursor protein described in the present invention is not limited to the above three subtypes.

[0029] The mutation type of the human amyloid precursor protein mutant may be, but is not limited to, at least one of a Swedish mutation, a Florida mutation, and a London mutation.

[0030] The amino acid sequence of the human presenilin 1 (PS1) is shown in SEQ ID NO.8, and the site where the mutation occurs in the human presenilin 1 mutant may be but is not limited to at least one of M146L / M146V, L286V, R278I, A246E, P264L, and deletion of the ninth exon.

[0031] Specifically, taking the construction of mouse models as an example, refer to the website Alzheimer's Disease Research Models | ALZFORUM or the literature (Esquerda-Canals G, et al. Mouse Models of Alzheimer's Disease. J Alzheimers Dis. 2017; 57(4): 1171-1183.) to select an AD model. The AD mouse model strains can be used but are not limited to: 3xTg, 5xFAD (B6SJL), 5xFAD (C57BL6), J20, A7 APP transgenic, ADanPP, AD-BXD, APP751SL / PS1 KI, APPswe / PSEN1dE9 (line 85), APPSwe / PSEN1dE9 (C3-3xS-9), APPswe / PSEN1dE9 (C57BL6), CAST.APP / PS1, etc.

[0032] In the construction method provided by the present invention, an animal model of Lewy body dementia is screened from the hybrid offspring of pink1 gene knockout animals and AD animals. Preferably, the target gene is detected by PCR method, and the screening method comprises: extracting offspring genomic DNA, using it as a template to identify whether the pink1 gene is knocked out and whether it contains Alzheimer's disease-related mutations by PCR method. Specifically, the pink1 gene, human amyloid precursor protein mutant gene, and human presenilin 1 mutant gene are identified, and offspring containing human amyloid precursor protein mutant gene and / or human presenilin 1 mutant gene and pink1 gene knockout are selected.

[0033] Preferably, the pink1 gene knockout identification primers are:

[0034] Upstream sequence: 5′-GCCAGAATGGGCTGTGGACAC-3′;

[0035] Downstream sequence: 5′-ACATGCAGTGAGCATGCAATGAG-3′;

[0036] APP gene identification primers are:

[0037] Upstream sequence: 5′-GACTGACCACTCGACCAGGTTCTG-3′;

[0038] Downstream sequence: 5′-CTTGTAAGTTGGATTCTCATATCCG-3′;

[0039] The primers for PS1 gene identification are:

[0040] Upstream sequence: 5′-AATAGAGAACGGCAGGAGCA-3′;

[0041] Downstream sequence: 5′-GCCATGAGGGCACTAATCAT-3′.

[0042] The present invention provides a Lewy body dementia animal model prepared by the above construction method.

[0043] The present invention also provides the use of the Lewy body dementia animal model prepared by the above construction method, including: using the model to screen or identify drugs that can prevent and alleviate α-synuclein aggregation associated with Lewy body disease.

[0044] Furthermore, the use includes: using the model to screen or identify drugs that can prevent, alleviate or treat Lewy body dementia, or to screen or identify biomarkers for early diagnosis of Lewy body dementia.

[0045] The present invention has the following beneficial effects:

[0046] (1) The method for constructing a novel animal model of Lewy body dementia provided by the present invention is to obtain offspring whose genomes have been knocked out of the pink1 gene and have mutations related to Alzheimer's disease by hybridizing pink1 gene knockout experimental animals with AD experimental animals simulating Aβ pathology. This is an animal model in which amyloid beta is deposited on the basis of pink1 gene knockout, α-synuclein protein aggregation is induced by β-amyloid plaques (Aβ), and Lewy bodies are widely produced in the cortex and hippocampus. This model can simulate the occurrence and development of DLB, provide a reliable animal model for disease mechanism research and therapeutic drug screening and development, and can also be combined with clinical practice to study and screen biomarkers that can diagnose Lewy body dementia at an early stage.

[0047] (2) The knocked-out and knocked-in genes in the construction method provided by the present invention are integrated into the genome and can be stably passed on to offspring through hybridization, thereby ensuring the stability of the model. The animal models obtained by screening exhibit behavioral and pathological phenotypes of DLB, and these phenotypes are aggravated with age. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] Figure 1 To construct pink1 gene knockout mice based on CRISPR-Cas9 technology, including: A. sgRNA target site; B. knockout mouse pink1 gene sequence compared with wild type (knockout site); C. knockout mouse PINK1 partial mRNA sequence and protein partial sequence compared with wild type.

[0049] Figure 2 The PINK1 protein of pink1 gene knockout mice is compared with the domain structure of the PINK1 protein of wild-type mice.

[0050] Figure 3 This is the breeding strategy of APP / PS1 mice with pink1 gene knockout, including: A. Schematic diagram of mice with different genotypes; B. Breeding strategy of pink1 gene knockout APP / PS1 mice (DLB model mice); C. PCR detection results of pink1 gene; D. PCR detection results of APP gene; E. PCR detection results of PS1 gene.

[0051] Figure 4 For young APP / PS1::pink1 - / -Mice showed short-term memory impairment in the Y maze, including: Schematic diagram of the AY maze test; Representative heat maps of the two stages of the BY maze test, the first row represents the training stage, two arms are opened, and mice are allowed to explore freely for 10 minutes, the second row represents the test stage, and a new arm is opened, and mice are allowed to explore freely for 10 minutes. In this stage, more mice enter the new arm, indicating that the mice have normal cognitive abilities; C. The percentage of the number of times they enter the two open arms during the training period; D. The percentage of the number of times they enter the three open arms during the test period, the white column corresponds to the Start arm; the gray column corresponds to the Another arm, and the yellow column corresponds to the novel arm. **P<0.01, ***P<0.001, comparison of the number of times they enter the three arms, One-way ANOVA test.

[0052] Figure 5 Comparison of mortality of four genotypes of mice before 5 months of age, APP / PS1::pink1 - / - The survival time of mice was significantly shortened.

[0053] Figure 6 The fecal water content test results are shown in Figure 2, where the fecal water content of the four genotype mice before death was compared with the average value of the previous test. - / - The water content of mice's feces decreased significantly.

[0054] Figure 7 For APP / PS1::pink1 - / - The distribution and pathological characteristics of Lewy bodies in the brain. A. From left to right: APP / PS1::pink1 - / - Typical images of α-syn and Aβ co-staining at three different locations in the mouse brain, showing that Lewy bodies are located in APP / PS1::pink1 - / - Widely distributed in the mouse cortex and hippocampus; B.APP / PS1::pink1 + / - and APP / PS1::pink1 - / - Pathological characteristics of Lewy bodies in the brain; C. Lewy bodies are distributed in APP / PS1::pink1 - / - In neurons of the mouse cortex, substantia nigra, VTA, and brainstem. DETAILED DESCRIPTION

[0055] The present invention will be further described below in conjunction with specific embodiments. The following embodiments are only used to illustrate the present invention and are not intended to limit the scope of application of the present invention. Without departing from the spirit and essence of the present invention, modifications or replacements made to the inventive method, steps or conditions all belong to the scope of the present invention.

[0056] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods; the materials and reagents used are reagents and materials that can be obtained from commercial channels unless otherwise specified.

[0057] The C57BL / 6J mice and APP / PS1 mice used in the following examples were purchased from Hangzhou Ziyuan Experimental Animal Technology Co., Ltd. APP / PS1 mice are a commercial model mouse widely used in AD research.

[0058] Background information of APP / PS1 mice: The APP gene is located on chromosome 21q11.2-22.2 and consists of 18 exons and 17 introns. The APP protein encoded by APP can be processed and hydrolyzed to produce β-amyloid protein. Under pathological conditions, APP is decomposed by β-secretase and γ-secretase to produce Aβ40 and Aβ42, of which Aβ42 is closely related to Aβ deposition and neuronal degeneration in the brain of AD patients. APP mutations can change the processing of APP, leading to the production of neurotoxic Aβ42, triggering a variety of pathological mechanisms, promoting neuronal apoptosis or death, and ultimately leading to the occurrence of AD. The PS1 gene is located on chromosome 14. The PS protein encoded by the PS1 gene is an important component of γ-secretase and plays an important role in the production of Aβ. PS1 mutation is one of the main causes of familial AD. PS1 mutation causes the hydrophilic loop domain of the protein it encodes to be missing, resulting in a change in its conformation, which can affect the activity of γ-secretase and increase the production of Aβ42.

[0059] Method for making APP / PS1 mouse model: The APP / PS1 double transgenic mouse model is bred by hybridizing PrP-hAPPK595N / M596L single transgenic dementia model mice and PrP-hPS1dE9 single transgenic dementia model mice.

[0060] Pathological manifestations of the brain of APP / PS1 mice: APP / PS1 transgenic mice developed senile plaques in the cerebral cortex at 4 and 5 months of age. The number and volume of senile plaques in the cerebral cortex increased significantly with age. Senile plaques similar to those of AD patients appeared in the brains of 9-12 month old mice.

[0061] Example 1: Construction of DLB ​​mouse animal model

[0062] Step 1: Construction of pink1 knockout mice based on CRISPR-Cas9 technology (pink1 - / - )

[0063] 1. sgRNA design and vector construction

[0064] According to the pink1 gene sequence of NCBI gene accession number Gene ID: 68943, two sgRNAs (1 and 2) were designed and selected through the E-CRISP Design website. The target sites of the two sgRNAs are as follows Figure 1 As shown in A. The sgRNA sequence is:

[0065] sgRNA1: 5'-GACAGCCATCTGCAGAGAGG-3';

[0066] sgRNA2: 5'-GCAGGCAGGACTCACCTCAG-3'.

[0067] Four oligos of two sgRNAs were synthesized by GenScript Biotech, and the two sgRNA single strands were complemented by annealing and cloned between the BbsI sites of pX330-U6-Chimeric_BB-CBh-hSpCas9.

[0068] 2. Construction of pink1 knockout mice using CRISPR-Cas9 technology

[0069] The Cas9 fragment and sgRNA fragment with T7 promoter were synthesized by PCR, and Cas9 was transcribed in vitro using MMESSAGE MMACHINE T7ULTRATranscription Kit.

[0070] mRNA, Guide-it sgRNA In Vitro Transcription Kit in vitro transcribes sgRNA,

[0071] RNAprep pure Tissue Kit was used to purify and concentrate RNA. The quality and concentration were checked by agarose gel electrophoresis and NanoDrop.

[0072] The purified cas9 mRNA and sgRNA were injected into the pronuclei of C57BL / 6J mice to obtain F0 generation mice.

[0073] The F0 generation mice were sequenced, and the sequencing results of the pink1 gene knockout mouse cDNA were compared with those of the wild-type mouse cDNA. Figure 1 As shown in B, PINK1 protein was expressed for comparison. Figure 1 As shown in C.

[0074] Comparison of the domain structure of PINK1 protein from pink1 knockout mice and wild-type mice ( Figure 2) and found that the PINK1 protein of pink1 knockout mice terminated at the S373 site, followed by an incorrect sequence of 12 amino acids.

[0075] After breeding the F0 generation mice, the F1 generation PINK1 gene knockout mice were obtained.

[0076] Step 2: Cross the pink1 knockout mice with the APP / PS1 mice to obtain APP / PS1::pink1 - / - Mouse

[0077] pass Figure 3 The mouse hybridization method shown in A and B of the present invention was used to purchase APP / PS1 mice from Hangzhou Ziyuan Experimental Animal Technology Co., Ltd. and cross them with the constructed pink1 - / - The mice were hybridized to obtain the F1 generation, and the genotype of the F1 generation was identified as APP / PS1::pink1 + / - Mice used for pink1 + / - orpink1 - / - APP / PS1::pink1 - / - Mice, according to Figure 3 The results of CE were evaluated to obtain pink1 gene knockout APP / PS1 mice (APP / PS1::pink1 - / - Mouse), the specific method of gene identification is as follows:

[0078] Cut the mouse tail or toes, add mouse tail lysis buffer and proteinase K, and lyse in a 55°C oven overnight; the next day, vortex and centrifuge, take the supernatant and add an equal amount of isopropanol, discard the supernatant, carefully wash with 70% ethanol, discard the supernatant, add ddH2O, put in a 55°C oven to mix, and use as a PCR template.

[0079] Table 1. PCR reaction system

[0080]

[0081] The upstream sequence of the primer for pink1 gene identification is: 5'-GCCAGAATGGGCTGTGGACAC-3'; the downstream sequence of the pink1 primer is: 5'-ACATGCAGTGAGCATGCAATGAG-3'.

[0082] Genotype pink1 + / - In the results, there was a band at 887 bp and 1021 bp respectively.

[0083] Table 2. PCR amplification process of pink1 gene fragment

[0084]

[0085] The upstream sequence of the primer for APP gene identification is: 5'-GACTGACCACTCGACCAGGTTCTG-3'; the downstream sequence of the APP primer is: 5'-CTTGTAAGTTGGATTCTCATATCCG-3'.

[0086] The upstream sequence of the primer for PS1 gene identification is: 5'-AATAGAGAACGGCAGGAGCA-3'; the downstream sequence of PS1 primer is: 5'-GCCATGAGGGCACTAATCAT-3'.

[0087] Table 3. The PCR amplification process of APP and PS1 gene fragments is as follows:

[0088]

[0089] Example 2: APP / PS1::pink1 - / - The consistency of clinical manifestations and pathology between mice and clinical DLB patients

[0090] 1. Dementia phenotype: young APP / PS1::pink1 - / - Mice showed short-term memory impairment in the Y-maze. The experimental method is as follows: The Y-maze has 3 arms (300mm×100mm×150mm), with patterns of different colors and shapes pasted inside the arms, which are divided into the start arm, other arm and novel arm. Experimental setting APP / PS1::pink1 - / - 、pink1 - / - , APP / PS1, and WT groups. The experiment was divided into two stages. In the training stage, the novel arm was blocked with a partition, and 2-3 month-old mice were placed from the starting arm. The camera was immediately turned on to record the free activities of the mice within 10 minutes. After 2 hours, the test stage was carried out. The partition was removed, and the mice were placed from the starting arm. The camera was immediately turned on to record the mice's stay time, number of entries, and movement trajectory in each arm within 10 minutes. Figure 4 A).

[0091] The results showed that there was no significant difference in the number of explorations of the two arms by the mice in each group during the training phase ( Figure 4 B, C) In the test phase after 2 h, WT mice and pink1 - / - and APP / PS1 mice showed a significant increase in the number of explorations of the new arm, while APP / PS1::pink1 - / - Mice showed no obvious tendency to explore the new arm ( Figure 4 B, D).

[0092] The above results show that pink1 - / -and APP / PS1 mouse models showed no damage at 2-3 months of age, while APP / PS1::pink1 - / - The mice developed impaired working memory at an early age and showed signs of dementia.

[0093] 2. Other clinical manifestations

[0094] (1) Decreased survival rate

[0095] The experimental mice were raised in the SPF-grade Animal Experiment Center of Zhejiang University, with the temperature controlled at 20-26℃ and 12h of sunshine. They were allowed to eat freely, and all feeding methods followed the Animal Welfare and Ethical Code of Zhejiang University. Under the above feeding conditions, the time of mouse death was found, and the age of the mouse at that time was recorded as the survival time. Each descending point on the statistical table was the time when a mouse died.

[0096] The results showed that APP / PS1 mice could survive for more than one year, and pink1 - / - The survival time of mice before 18 months of age is similar to that of wild-type mice. - / - Mice can experience sudden death at around 2 months of age. The dead mice usually have shiny fur and no obvious weight loss before death. The overall survival rate is about 20% ( Figure 5 ).

[0097] (2) Decreased fecal water content

[0098] Fecal water content is an indicator for detecting constipation in mice. The fecal water content of mice was measured once a week. The last measured value was compared with the average value of the previous multiple tests. It was found that wild-type mice, pink1 - / - There was no significant difference in fecal water content between APP / PS1 mice and APP / PS1 mice, whereas APP / PS1::pink1 - / - Mice showed a significant decrease ( Figure 6 ), which may be caused by abnormal function of the enteric nervous system.

[0099] Example 3: APP / PS1::pink1 - / - Mice and APP / PS1::pink1 + / - Lewy body formation in mice

[0100] 1. Frozen sections

[0101] The mouse brain tissue was obtained for whole-brain sectioning using a cryostat (Leica CM 1900, Leica Corporation, USA) with a section thickness of 25 μm / slice. A brain slice was taken every 200 μm for subsequent staining.

[0102] 2. Immunofluorescence co-staining

[0103] 1) Place the brain slices in PBS and wash them three times with PBS on a shaker;

[0104] 2) Aspirate and discard PBS, add 0.3% Triton-X PBS, and place on a shaker at room temperature to break the membrane for 30 minutes;

[0105] 3) Wash with PBS at room temperature on a shaker for 5 minutes and repeat three times;

[0106] 4) Aspirate and discard PBS, add blocking solution (Biyuntian, product number P0260, QuickBlock TM Immunostaining blocking solution), incubated on a shaker at room temperature for 2 h;

[0107] 5) Add primary antibodies (α-syn: CST, Catalog No. D37A6, rabbit, dilution ratio 1:600; Aβ: Biolegend, Catalog No. 803001S, mouse, dilution ratio 1:300; Bio-Tech, Catalog No. P0262, QuickBlock TM Immunostaining primary antibody dilution solution), incubated in a 4°C refrigerator overnight;

[0108] 6) Remove the primary antibody and wash on a shaking table with PBS for 5 minutes, repeat 3 times;

[0109] 7) Remove PBS and add secondary antibody (Biyuntian, Cat. No. P0108, QuickBlock TM Immunostaining primary antibody diluent; Jackson, catalog number 015-090-050, Fluorescein (FITC)-conjugated ChromPure Mouse Transferrin, 1:200; Jackson, catalog number 011-160-003, Cy3-conjugated ChromPure Rabbit IgG, 1:200), incubate at room temperature on a shaker for 2 hours.

[0110] 8) Remove the secondary antibody and wash with PBS three times, 5 minutes each time;

[0111] 9) Spread the brain slices flat on a glass slide, remove excess PBS with a syringe, and let them dry;

[0112] 10) Seal the slides with a sealing medium (Biyuntian, P0131, anti-fluorescence quenching sealing solution (containing DAPI)).

[0113] Using immunofluorescence colocalization, we found that APP / PS1::pink1 - / - Mice and APP / PS1::pink1 + / - The mouse brain contains Lewy bodies formed by α-Syn aggregation, which are widely distributed in the cortex and hippocampus ( Figure 7 A). There are two main pathological features of Lewy bodies. The first is a "jewel flower"-like Lewy body, usually with an Aβ plaque in the middle and α-Syn in a "dot-like" pattern surrounding the Aβ plaque, suggesting that it is a Lewy neurite ( Figure 7 B); the second is the distribution of Lewy bodies in neurons, which can be distributed in neurons of the cortex, substantia nigra, VTA, and brainstem ( Figure 7 C).

[0114] In pink1 - / - No Lewy bodies were found in the mouse hippocampus; Aβ plaques but no Lewy bodies were formed in the cortex of APP / PS1 mice.

[0115] Example 4: APP / PS1::pink1 - / - Stability of mouse model

[0116] use Figure 3 The hybridization method shown was used to crossbreed the F1 generation mice and their offspring. All offspring were identified by PCR of pink1, PS1, and APP genes one month after birth, and APP / PS1::pink1 was obtained. - / - Mouse.

[0117] The results show that all APP / PS1::pink1 - / - Mice can develop behavioral and pathological phenotypes of DLB ​​around 4 months of age, and some APP / PS1::pink1 + / - Mice also develop behavioral and pathological phenotypes of DLB, which worsen with age.

[0118] Since the model constructed by the present invention is a transgenic model, these knocked-out and knocked-in genes can be stably passed on to offspring, thus ensuring the stability of the model.

[0119] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions or improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention. For example, in the selection of AD models, existing commercial AD mice that can simulate Aβ pathology can be hybridized with pink1 gene knockout mice to establish a Lewy body dementia model.

Claims

1. A method for constructing an animal model of Lewy body dementia, characterized in that: The following steps are involved: (1) Using gene editing technology, the expression of the phosphatase and tensin homologous gene-induced kinase 1 gene pink1 in the genome of the experimental animal is deleted to obtain pink1 gene knockout homozygous or heterozygous experimental animal A; (2) Crossbreeding the pink1 gene knockout homozygous or heterozygous experimental animal A with an Alzheimer's disease model experimental animal B, wherein the Alzheimer's disease model experimental animal has an Alzheimer's disease-associated mutation in its genome and exhibits β-amyloid plaque pathology, to obtain offspring, from which offspring containing both the Alzheimer's disease-associated mutation and the pink1 gene knockout heterozygous or homozygous are screened, thereby obtaining the Lewy body dementia animal model.

2. The method for constructing an animal model of Lewy body dementia according to claim 1, characterized in that: The experimental animal A and the experimental animal B are one of mice, rats, guinea pigs and macaques.

3. The method for constructing an animal model of Lewy body dementia according to claim 2, characterized in that: In step (1), the pink1 gene in the experimental animal genome is knocked out using CRISPR-Cas9 technology, the CDS sequence of the mouse pink1 gene is shown in SEQ ID NO.1, the CDS sequence of the rat pink1 gene is shown in SEQ ID NO.2, the CDS sequence of the guinea pig pink1 gene is shown in SEQ ID NO.3, and the CDS sequence of the macaque pink1 gene is shown in SEQ ID NO.

4.

4. The method for constructing an animal model of Lewy body dementia according to claim 3, characterized in that: For the knockout of mouse pink1 gene, a pair of sgRNA sequences were designed, and their nucleotide sequences were 5'-GACAGCCATCTGCAGAGAGG-3' and 5'-GCAGGCAGGACTCACCTC AG-3'.

5. The method for constructing an animal model of Lewy body dementia according to claim 1, characterized in that: The experimental animal B is a transgenic animal that overexpresses a human amyloid precursor protein mutant and / or a human presenilin 1 mutant in animal neurons.

6. The method for constructing an animal model of Lewy body dementia according to claim 1, characterized in that: The experimental animal B adopts any one of the mouse models 3xTg, 5xFAD, J20, A7 APP transgenic, ADanPP, AD-BXD, APP751SL / PS1 KI, APPswe / PSEN1dE9, and CAST.APP / PS1.

7. The method for constructing an animal model of Lewy body dementia according to claim 1, characterized in that: In step (2), the method for screening an animal model of Lewy body dementia includes: extracting genomic DNA of offspring and using it as a template to identify whether the pink1 gene is knocked out and whether it contains Alzheimer's disease-related mutations using a PCR method.

8. The method for constructing an animal model of Lewy body dementia according to claim 7, characterized in that: The primers for pink1 gene knockout identification are: Upstream sequence: 5′-GCCAGAATGGGCTGTGGACAC-3′; Downstream sequence: 5′-ACATGCAGTGAGCATGCAATGAG-3′; The primers for identifying human amyloid precursor protein mutant genes are: Upstream sequence: 5′-GACTGACCACTCGACCAGGTTCTG-3′; Downstream sequence: 5′-CTTGTAAGTTGGATTCTCATATCCG-3′; The primers for identifying human presenilin 1 mutant gene are: Upstream sequence: 5′-AATAGAGAACGGCAGGAGCA-3′; Downstream sequence: 5′-GCCATGAGGGCACTAATCAT-3′.

9. Use of the Lewy body dementia animal model prepared by the construction method according to any one of claims 1 to 8, characterized in that: include: This model is used to screen or identify drugs that can prevent or alleviate α-synuclein aggregation associated with Lewy body disease.

10. Use of the Lewy body dementia animal model prepared by the construction method according to any one of claims 1 to 8, characterized in that: include: The model is used to screen or identify drugs that can prevent, alleviate or treat Lewy body dementia, or to screen or identify biomarkers for early diagnosis of Lewy body dementia.

Images