A method for constructing an animal model of Lewy body dementia and its uses

Abstract

The present invention discloses a method for constructing and using an animal model of dementia with Lewy bodies, and belongs to the technical field of animal model construction. The construction method comprises: obtaining a phosphatase and tensin homologous gene-induced kinase 1 gene knockout experimental animal A using gene editing technology, and crossbreeding with an Alzheimer's disease model experimental animal B simulating β-amyloid protein (Aβ) pathology, and screening offspring containing both Alzheimer's disease-related mutations and pink1 gene knockout. The model constructed by the present invention is to deposit β-amyloid protein on the basis of pink1 gene knockout, induce α-synuclein protein aggregation using β-amyloid plaques (Aβ), widely produce Lewy bodies in the cortex and hippocampus, simulate the occurrence and development process of DLB, and provide a reliable animal model for disease mechanism research and therapeutic 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 an animal model of Lewy body dementia. Background Art

[0002] Dementia with Lewy bodies (DLB) is a neurodegenerative disease, the second most common type of degenerative dementia after Alzheimer's disease (AD), accounting for approximately 10-15% of dementia cases. Key clinical manifestations include Parkinson's disease (PD)-like symptoms, such as cognitive impairment, visual hallucinations, rapid eye movement (REM) sleep disturbances, and bradykinesia. These symptoms may be accompanied by digestive system symptoms such as constipation and cardiovascular symptoms such as arrhythmias. Imaging studies using positron emission tomography (PET) can reveal decreased dopamine transport in the basal ganglia. Characteristic pathological changes are the formation of Lewy bodies, primarily composed of α-synuclein, in the brain. These Lewy bodies are primarily distributed throughout the cortex.

[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 it and many other diseases such as Parkinson's disease, sleep disorders, digestive system abnormalities, schizophrenia, and Alzheimer's disease. In addition, due to the possibility of arrhythmia, it can easily 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. For example, acetylcholinesterase inhibitors, which are used to treat Alzheimer's disease, are used to treat dementia symptoms.

[0004] Therefore, there is an urgent need in this field to study the pathogenesis of DLB, diagnostic methods, and 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 a DLB model, the animal model of Parkinson's disease has been "borrowed" mainly because Parkinson's disease also forms Lewy bodies due to α-synuclein protein aggregation. DLB and PD are both 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 of 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] The models currently used for DLB research are mainly transgenic mouse models and cell models. Mouse models usually require mice that overexpress α-synuclein protein, combined with specific protein knockout or overexpression, and finally require the 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" and adds it to primary cultured rat or mouse neurons to observe the aggregation of α-synuclein protein in inclusion bodies within neurons. This cell model has also been used in PD research.

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

[0008] Effective animal models are crucial tools for studying disease mechanisms and therapeutic agents. Ideally, a DLB model should not overexpress α-synuclein, possess gene disruption detectable in clinical patients, and exhibit a dementia phenotype that partially or fully mimics the clinical manifestations of DLB, such as irritability, constipation, and sudden death. Crucially, pathological examination should reveal widespread Lewy bodies in the cortex and hippocampus. However, to date, no animal model has been developed that accurately simulates the onset and progression of DLB, hindering in-depth research. Therefore, establishing an animal model of DLB ​​that demonstrates both early phenotypes and a consistent progression is crucial. 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 of Lewy body dementia, and 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 solutions:

[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, and pink1 gene knockout homozygotes (pink1 - / - ) or heterozygotes (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 that simulate β-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, its clinically relevant phenotypes include: (1) early sudden death; (2) constipation and difficulty defecating; (3) dilated cardiomyopathy; (4) dementia; (5) neurological damage; (6) extensive Lewy bodies in the brain; and (7) Lewy bodies in the intestine and heart.

[0017] The offspring can be the first generation obtained by hybridizing the parents; or the nth generation obtained by hybridizing the offspring with the offspring or with the parents, where n ≥ 2. For example, the F1 generation is obtained by hybridizing the parents, and the F1 generation is screened for offspring individuals that contain both an Alzheimer's disease-associated mutation and a pink1 gene knockout heterozygote to obtain the Lewy body dementia animal model. Alternatively, an F1 generation offspring individual containing both an Alzheimer's disease-associated mutation and a pink1 gene knockout heterozygote may be bred with an offspring individual containing only a pink1 gene knockout heterozygote to produce an F2 generation; alternatively, an F1 generation offspring individual containing both an Alzheimer's disease-associated mutation and a pink1 gene knockout heterozygote may be bred with a parental pink1 gene knockout homozygote or pink1 gene knockout heterozygote to produce an F2 generation; alternatively, two F1 generation offspring individuals containing both an Alzheimer's disease-associated mutation and a pink1 gene knockout heterozygote may be bred with an F2 generation, and then the F2 generation may be screened for offspring individuals containing both an Alzheimer's disease-associated mutation and a pink1 gene knockout heterozygote or homozygote to produce the Lewy body dementia animal model. The F2 generation may then be bred in the same manner as above.

[0018] Furthermore, the experimental animals used to construct the Lewy body dementia animal model of the present invention can be experimental animals commonly used in research in this 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 nuclease.

[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; and 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 sites. All 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 herein is an animal model obtained through genetic engineering techniques that harbors mutations associated with Alzheimer's disease and exhibits β-amyloid plaque pathology. This experimental animal can be a commercially available AD animal model or constructed based on literature reports.

[0026] The mutations associated with Alzheimer's disease include, but are not limited to, mutations in the amyloid precursor protein (APP) gene and / or presenilin 1 gene sequence. The present invention can also employ 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 its neurons. The transgenic animal overexpresses mutant human amyloid precursor protein and human presenilin 1 in its neurons, resulting in an increase in the longer form of beta-amyloid protein (the main component of amyloid protein deposits found in the brains of AD patients).

[0028] The human amyloid precursor protein has three common isoforms: 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 isoforms.

[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, for example, the construction of a mouse model can be used to select an AD model by referring 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). AD mouse model strains can be used, but are not limited to, the following: 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, the human amyloid precursor protein mutant gene, and the human presenilin 1 mutant gene are identified, and offspring containing the human amyloid precursor protein mutant gene and / or the human presenilin 1 mutant gene and the pink1 gene knockout are selected.

[0033] Preferably, the primers for identifying pink1 gene knockout 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-mentioned construction method, including: using the model to screen or identify drugs that can prevent or 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 in which the pink1 gene is knocked out and contains Alzheimer's disease-related mutations by hybridizing pink1 gene knockout experimental animals with AD experimental animals simulating Aβ pathology. This is an animal model in which beta-amyloid protein is deposited on the basis of pink1 gene knockout, beta-amyloid plaques (Aβ) are used to induce α-synuclein protein aggregation, and Lewy bodies are widely produced in the cortex and hippocampus. This model can simulate the occurrence and development process of DLB, providing a reliable animal model for disease mechanism research and therapeutic drug screening and development. It can also be combined with clinical research to study and screen biomarkers that can diagnose Lewy body dementia at an early stage.

[0047] (2) The knockout and knock-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 hybrid breeding, 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 Construction of pink1 knockout mice based on CRISPR-Cas9 technology, including: A. sgRNA target site; B. Comparison of the pink1 gene sequence of the knockout mouse with that of the wild type (knockout site); C. Comparison of the partial mRNA and protein sequences of PINK1 in the knockout mouse with those of the wild type.

[0049] Figure 2 The PINK1 protein from pink1 gene knockout mice is compared with the domain structure of the PINK1 protein from 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 exhibit short-term memory impairment in the Y-maze test. Figures: Schematic diagram of the Y-maze test; Representative heatmaps from two phases of the BY-maze test. The first row represents the training phase, in which mice were allowed to freely explore both arms for 10 minutes. The second row represents the test phase, in which mice were allowed to freely explore a new arm for 10 minutes. During this phase, mice entered the new arm more frequently, indicating normal cognitive ability. C. Percentage of entries into the two open arms during the training phase; D. Percentage of entries into the three open arms during the test phase. White bars correspond to the Start arm; gray bars correspond to the Another arm; and yellow bars correspond to the Novel arm. **P < 0.01, ***P < 0.001, comparison of entries into the three arms, one-way ANOVA.

[0052] Figure 5 Comparison of mortality of four genotype 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 as follows: the fecal water content of the four genotype mice before death was compared with the average value of the previous test, APP / PS1::pink1 - / - The water content of mice's feces decreased significantly.

[0054] Figure 7 For APP / PS1::pink1 - / - 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 Lewy bodies 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 examples. The following examples 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 fall within the scope of the present invention.

[0056] Unless otherwise specified, the experimental methods used in the following examples are conventional methods; the materials and reagents used are commercially available reagents and materials 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 on 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 undergoes proteolysis to produce amyloid-β. In pathological conditions, APP is broken down by β-secretase and γ-secretase to produce Aβ40 and Aβ42. Aβ42 is closely associated with Aβ deposition and neuronal degeneration in the brains of patients with AD. APP mutations can alter APP processing, leading to the production of the neurotoxic Aβ42, triggering various pathological mechanisms that promote neuronal apoptosis or death, ultimately leading to AD. The PS1 gene, located on chromosome 14, encodes the PS protein, a crucial component of γ-secretase and plays a crucial role in Aβ production. PS1 mutations are a major cause of familial AD. PS1 mutations result in a loss of the hydrophilic loop domain of the encoded protein, altering its conformation and impairing γ-secretase activity, leading to increased Aβ42 production.

[0059] Method for preparing the APP / PS1 mouse model: The APP / PS1 double transgenic mouse model was 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 develop senile plaques in the cerebral cortex at 4 and 5 months of age. As the age increases, the number and volume of senile plaques in the cerebral cortex increase significantly. Senile plaques similar to those of AD patients appear in the brains of 9-12 month old mice.

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

[0062] Step 1: Construction of pink1 gene 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 complementary by annealing and cloned between the BbsI sites of pX330-U6-Chimeric_BB-CBh-hSpCas9.

[0068] 2. Construction of pink1 gene 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 transcription of sgRNA,

[0071] RNA was purified and concentrated using the RNAprep pure Tissue Kit. Quality and concentration were assessed 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 pink1 gene knockout mouse cDNA were compared with those of 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 gene 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 - / - mice

[0077] pass Figure 3 The mouse hybridization method shown in A and B was used to hybridize APP / PS1 mice purchased from Hangzhou Ziyuan Experimental Animal Technology Co., Ltd. and then hybridized 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 + / - or pink1 - / - 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 mix 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, and mix in a 55°C oven to 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 exhibit short-term memory impairment in a Y-maze. The experimental method is as follows: The Y-maze consists of three arms (300mm×100mm×150mm), each with patterns of different colors and shapes. These arms are divided into a start arm, an other arm, and a novel arm. Experimental setup: APP / PS1::pink1 - / - 、pink1 - / - The experiment was divided into two phases. In the training phase, the novel arm was blocked by a partition, and 2-3 month old mice were placed in the starting arm. The mice's free movement was immediately recorded for 10 minutes. After 2 hours, the test phase was carried out. The partition was removed and the mice were placed in the starting arm. The mice's stay time, number of entries, and movement trajectory in each arm were immediately recorded for 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 each group of mice 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 did not show a clear 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, whereas APP / PS1::pink1 - / - The mice developed impaired working memory at an early age and exhibited dementia.

[0093] 2. Other clinical manifestations

[0094] (1) Decreased survival rate

[0095] Experimental mice were housed in the SPF-grade animal laboratory center of Zhejiang University, with a temperature controlled at 20–26°C and 12 h of daylight. They were allowed to eat freely. All husbandry methods adhered to the Animal Welfare and Ethical Code of Zhejiang University. Under these husbandry conditions, the time of death of a mouse was recorded as the survival time, and each decreasing point on the statistical table represents the time of death of a mouse.

[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 die suddenly at around 2 months old. 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 measurement value was compared with the average value of the previous multiple measurements. It was found that wild-type mice and 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 due to abnormal function of the enteric nervous system leading to constipation.

[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 freezing microtome (Leica CM 1900, Leica, USA). The section thickness was 25 μm per slice, and a brain slice was taken every 200 μm for subsequent staining.

[0102] 2. Immunofluorescence co-staining

[0103] 1) Place brain slices in PBS and wash 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 for 30 minutes to disrupt the membrane;

[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) and incubated on a shaker at room temperature for 2 h;

[0107] 5) Add primary antibodies (α-syn: CST, product number D37A6, rabbit, dilution ratio 1:600; Aβ: Biolegend, product number 803001S, mouse, dilution ratio 1:300; Biyuntian, product number P0262, QuickBlock TM Immunostaining primary antibody dilution solution), incubated overnight at 4°C refrigerator;

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

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

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

[0111] 9) Spread the brain slice flat on a glass slide, remove excess PBS with a syringe, and let it dry.

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

[0113] Using immunofluorescence colocalization, we found that APP / PS1::pink1 - / - Mice and APP / PS1::pink1 + / - There are Lewy bodies formed by α-Syn aggregation in the mouse brain, 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 with an Aβ plaque in the middle and α-Syn in the form of "dots" surrounding the Aβ plaque, suggesting Lewy neurites ( Figure 7 B); secondly, Lewy bodies are distributed 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 hippocampus of mice; Aβ plaques but no Lewy bodies were formed in the cortex of APP / PS1 mice.

[0115] Example 4: APP / PS1::pink1 - / - Stability of the 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 ​​at 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 in 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 scope of protection 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 to modify the expression of phosphatase and tensin homolog gene-induced kinase 1 in the genome of experimental animals pink1 Loss of gene expression, gain pink1 Gene knockout homozygous or heterozygous experimental animals A; (2) pink1 A gene knockout homozygous or heterozygous experimental animal A is crossbred with an Alzheimer's disease model experimental animal B, wherein the experimental animal B is a transgenic animal that overexpresses a human amyloid precursor protein mutant and a human presenilin 1 mutant in the animal's neurons, and offspring are obtained, from which offspring are screened for offspring that simultaneously contain a human amyloid precursor protein mutation, a human presenilin 1 mutation, and pink1 The offspring of the gene knockout heterozygote or homozygote obtain the Lewy body dementia animal model; The experimental animals A and B are mice.

2. The method for constructing an animal model of Lewy body dementia according to claim 1, wherein: In step (1), CRISPR-Cas9 technology is used to knock out the pink1 Gene, mouse pink1 The CDS sequence of the gene is shown in SEQ ID NO.

1.

3. The method for constructing an animal model of Lewy body dementia according to claim 2, wherein: For mice pink1 For gene knockout, a pair of sgRNA sequences were designed, with the nucleotide sequences of 5'-GACAGCCATCTGCAGAGAGG-3' and 5'-GCAGGCAGGACTCACCTC AG-3'.

4. The method for constructing an animal model of Lewy body dementia according to claim 1, wherein: In step (2), the method for screening the animal model of Lewy body dementia includes: extracting genomic DNA of offspring, using it as a template and using PCR method to pink1 The gene was identified to determine whether it was knocked out and whether it contained human amyloid precursor protein mutation and human presenilin 1 mutation.

5. The method for constructing an animal model of Lewy body dementia according to claim 4, wherein: pink1 The primers for 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 genes are: Upstream sequence: 5′-AATAGAGAACGGCAGGAGCA-3′; Downstream sequence: 5'-GCCATGAGGGCACTAATCAT-3'.

6. Use of the Lewy body dementia animal model prepared by the construction method according to any one of claims 1 to 5, characterized in that: include: This model is used to screen or identify drugs that can prevent or alleviate Lewy body disease-associated α-synuclein aggregation.

7. Use of the Lewy body dementia animal model prepared by the construction method according to any one of claims 1 to 5, 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.

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