A method for constructing a mouse model that simulates the natural course of AD

Abstract

The application belongs to the field of biological medicine, and relates to a mouse model construction method of a mouse model simulating the natural pathogenesis process of AD. The Calnexin (CNX) gene is knocked down in the hippocampus of a healthy adult mouse, and the pathological process of simulating the natural pathogenesis of AD is realized. The expression amount of the CNX gene in the brain hippocampus of the mouse model is more than 40% lower than that of a wild-type mouse, and the mouse model is accompanied by A beta-like protein increase and NFT aggregation caused by Tau protein phosphorylation, and the mouse model shows pathological behavior characteristics consistent with AD mice in behavior.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of biological medicine, and relates to a method for constructing a mouse model simulating the onset process of AD. BACKGROUND

[0002] Alzheimer's disease (hereinafter referred to as "AD") is the most common neurodegenerative disease in the elderly, and its characteristic is memory dysfunction. In clinical practice, it mainly manifests as cognitive dysfunction, gradual decline in self-care ability and mental disorders. The main pathological features are senile plaques formed by intracellular aggregated beta amyloid (Aβ) and neurofibrillary tangles (NFTs) formed by extracellular deposited phosphorylated Tau protein. It has been found in research that the formation of beta amyloid (Aβ) aggregation and neurofibrillary tangles (NFTs) formed by Tau protein hyperphosphorylation in the pathological features of AD may be related to the dysfunction of Calnexin (CNX). CNX is a membrane-bound chaperone that assists in the folding, processing and quality control of nascent glycoproteins, together with other soluble endoplasmic reticulum lumen homologs, calnexin. Kraus et al. found that CNX-deficient mice can survive at birth, but have a high neonatal mortality rate and suffer from neurological diseases, including ataxia and peripheral neuropathy abnormalities, manifested as unsteady gait, hind limb extension, tremor and rolling walk.

[0003] According to the statistical data collected worldwide, the risk of disease gradually increases with age, and the incidence of AD doubles every 5 years after the age of 65. According to the data of WHO, it is estimated that the global AD patients will reach 5000 million by 2030. Among them, there will be about 1500 million AD patients in China, and the number is still increasing dramatically with the intensification of global aging. However, as of now, there is no effective cure for AD. Due to the incomplete understanding of the etiology and pathogenesis of AD, there is still no effective treatment method, and 99% of AD new drug development ends in failure in phase II / III.

[0004] In order to strengthen the clinical medical transformation research and preclinical drug screening, it is urgent to develop and develop related animal models. AD animal model is to simulate the pathological changes and behavioral characteristics of brain tissue of AD patients in experimental animals. At present, most AD animal models can only simulate some pathological changes or symptoms of AD, and animal models completely consistent with the onset process and pathological mechanism of human AD have not yet been reported. At present, the main AD mouse model construction methods are:

[0005] (1) APP transgenic mouse model: The human APP gene is integrated into the mouse genome, expressed, and inherited. Compared with normal animals, APP transgenic animals have excessive Aβ expression in their brains, causing cognitive impairment and other clinical pathological features of AD. Unlike AD patients, APP transgenic mice do not have NFT neuropathological changes, and most only have hyperphosphorylation of Tau protein.

[0006] (2) Double transgenic and triple transgenic mouse models: APP transgenic mice and Tau transgenic mice are mated to obtain double transgenic mice with APP / Tau overexpression; further mating with PSEN1 (presenilins 1) transgenic mice can produce APP / Tau / PSEN1 triple transgenic mice. Although the APP / Tau and APP / Tau / PSEN1 transgenic mouse models partially reproduce the following features: Aβ deposition and Tau hyperphosphorylation with the formation of NFT-like structures, the two-gene / three-gene AD model mice have low birth rates, difficulty in birth and culture, short lifespan, and significant differences in AD behavioral characteristics, which also indirectly indicates that there are significant differences between the model and the pathogenesis of human AD. (3) Fornix fimbria injury model: surgical methods are used to damage the projection of cholinergic nerve fibers in the septum to the hippocampus and cortex, thereby destroying cholinergic-induced learning and memory disorders in animals. After surgery, animals develop learning and memory dysfunction, but no Aβ and NFT lesions appear. The damage range is large, requiring the implementer to be very skilled to successfully construct the animal model, and it is difficult to mass-produce and sell the animal model.

[0007] At present, the AD mouse models available on the market in China are mainly APP mice, APP / Tau / mice and APP / Tau / PSEN1 mice. The Chinese patent "CN201510990290.3 A method for generating a HO-1 / APP / PSEN1 triple-transgenic Alzheimer's disease mouse model" discloses a HO-1 / APP / PSEN1 triple-transgenic mouse. The core technology of this patent is that HO-1 replaces Tau in APP / Tau / PSEN1. The constructed mice have not been compared with APP / Tau / PSEN1, and there are no obvious differences in lifespan and pathological characteristics.

[0008] In summary, the causes and pathogenesis of AD have not yet been fully revealed, and there has been almost no progress in the development of new drugs targeting Aβ and NFT. Therefore, the construction method of mouse models that can "simulate the natural pathogenesis of AD" plays a vital role in the study of AD mechanisms and as preclinical animal testing for drugs. Summary of the Invention

[0009] In view of the existing technical problems, the mouse model construction method of the mouse model simulating the natural onset process of AD is provided, the CNX gene in the hippocampus of a healthy adult mouse is knocked down, and the pathological process of simulating the natural onset of AD is realized.

[0010] Specifically, the technical solutions of the present application are as follows:

[0011] The first aspect of the present application provides a mouse model construction method of a mouse model simulating the natural onset process of AD, a recombinant vector (SYN-CNX-KD) carrying a CNX knockout gene is introduced into the hippocampus of a mouse brain by a minimally invasive method to complete the knockdown of the CNX gene of neurons, thereby realizing the simulation of the natural onset process of AD. The expression amount of the CNX gene in the hippocampus of the mouse model obtained by the method is more than 40% lower than that of a wild type mouse, accompanied by the aggregation of NFT caused by the increase of Aβ-like protein and the phosphorylation of Tau protein, and the pathological behavior characteristics of the mouse model are consistent with those of AD mice in behavior.

[0012] The second aspect of the present application provides a vector system for knocking out the target gene CNX gene, including adenovirus, adeno-associated virus, lentivirus, Cre-Lox and CRISPR-Cas system.

[0013] The third aspect of the present application provides a construction method of a CNX-D model mouse, and the specific steps include:

[0014] 1. Cloning the upstream and downstream recombinant homologous fragments of the CNX gene, respectively, and enzyme cutting and linking;

[0015] 2. Connecting the cloned fragment of 1 into a vector to form a pSYN-CNX-KO knockout vector;

[0016] 3. Preparing a DNA solution for minimally invasive injection;

[0017] 4. Injecting the constructed recombinant vector into the hippocampus of a mouse brain by a minimally invasive injection method, and screening a positive clone.

[0018] In the present application, the identification method of the obtained mouse model includes molecular detection related to AD disease, pathological analysis, immunohistochemical analysis and observation of behavioral characteristics.

[0019] Compared with the prior art, the present application has the following advantages and beneficial effects:

[0020] 1. The mouse model construction method provided by the present application simulates the natural onset process of AD, which has a short time, good reproducibility and high positive success rate (the expression amount of the CNX gene is reduced by more than 40% after 1 month of knockdown, and the positive rate is more than 80%) compared with the artificial aging method.

[0021] 2. The mouse model constructed by the application has no significant difference in signs and lifespan from wild type, and the pathological progression process is more natural (generally after 1 month, similar behavioral characteristics to APP mice appear).

[0022] 3. Compared with surgical physical methods, the treatment method is more gentle, has strong target area specificity, low off-target probability, and almost no effect on other brain regions of non-target regions, effectively excludes other factors interference, and is very suitable for pathological basic research and preclinical research of drug development. BRIEF DESCRIPTION OF DRAWINGS

[0023] The drawings described herein are used to provide further understanding of the embodiments of the application, constitute a part of the application, and do not constitute a limitation of the embodiments of the application.

[0024] Figure 1 Schematic diagram of CNX gene knockout.

[0025] Figure 2 APP mouse and normal mouse hippocampus CNX protein expression analysis, wherein: A and B are WB detection of CNX protein expression in mouse hippocampus and analysis; C and D are immunofluorescence detection of CNX protein expression in mouse hippocampus, Bar = 200 μm. Data are expressed as mean ± standard error of mean (SEM) (*P<0.05, **P<0.01, ***P<0.001).

[0026] Figure 3 Normal mouse hippocampus CNX knockdown causes cognitive dysfunction in mice, wherein: A is a schematic diagram of the experimental process; B is a hippocampal stereotactic injection and fluorescence GFP effect diagram; C is the escape latency of two groups of mice detected in the Morris water maze for 6 consecutive days; D and E are WB detection of CNX protein expression difference in hippocampus tissue of two groups of mice; F and G are swimming speed and escape latency on the first day of the Morris water maze; H, I and J are the distance, time and number of times of crossing the platform in the target quadrant; K is the mouse trajectory diagram; L and M are the cognitive coefficient and discrimination coefficient of the mouse novel object recognition experiment.

[0027] Data are expressed as mean ± standard error of mean (SEM) (*P<0.05, **P<0.01, ***P<0.001).

[0028] Figure 4CNX knockdown in normal mouse hippocampus caused damage to mouse neurons, wherein: A and C were TUNEL detection of mouse apoptosis and analysis, Bar = 200 μm; B and D were FJC detection of mouse neuron death and analysis, Bar = 200 μm; E and H were Nissl staining detection of mouse neuron damage and analysis, Bar = 25 μm; F, G and I were WB detection of NeuN and PSD95 protein expression levels in mouse hippocampus. Data were expressed as mean ± standard error of mean (SEM) (*P<0.05, **P<0.01, ***P<0.001).

[0029] Figure 5 CNX knockdown in normal mouse hippocampus increased Tau protein phosphorylation and caused Aβ level changes, wherein: A, B, C and D were WB detection of Tau protein phosphorylation level; E and F were ELISA detection of soluble and insoluble Aβ1-40 protein levels; G and H were ELISA detection of soluble and insoluble Aβ1-42 protein levels; H and I were thioflavin S detection of amyloid plaque levels, Bar = 200 μm. Data were expressed as mean ± standard error of mean (SEM) (*P<0.05, **P<0.01, ***P<0.001).

[0030] Figure 6 CNX overexpression in APP mouse hippocampus improved the cognitive function of AD mice, wherein: A was the co-localization of neuron MAP2, astrocyte GFAP, microglia IBA1 markers and virus tag Flag, Bar = 50 μm; B and C were WB detection of CNX protein expression in hippocampus; D and E were immunofluorescence detection of CNX protein expression in hippocampus, Bar = 50 μm; F was the escape latency of Morris water maze for 5 consecutive days; G was the mouse trajectory map; H, I and J were the distance, time and number of crossing the platform in the target quadrant; K and L were the swimming speed and escape latency of the first day of Morris water maze; M and N were the cognitive coefficient and discrimination coefficient of the novel object recognition experiment. Data were expressed as mean ± standard error of mean (SEM) (*P<0.05, **P<0.01, ***P<0.001). DETAILED DESCRIPTION

[0031] In order to make the purpose, technical scheme and advantages of the present application clearer, further detailed description of the present application will be given below in combination with examples and drawings, the illustrative embodiments of the present application and the description thereof are only used to explain the present application, and do not limit the present application.

[0032] Example 1: Comparison of CNX expression amount in normal mice and APP mice

[0033] To determine whether CNX expression changes in the brain under AD disease conditions, normal mice (C57BL / 6J) and AD (APP / PS1) mice were selected as research subjects, and hippocampal tissue was extracted from mice and high-throughput transcriptome sequencing analysis was performed on the hippocampal tissue. The experimental results showed that compared with normal mice, the CNX gene was downregulated in AD mice, and the CNX protein in the hippocampus of AD mice was reduced by about 30-50% ( Figure 2 The expression of CNX in the brain of AD mice was verified by immunofluorescence using a mouse primary antibody against CNX (1:200). The results of staining with 647 mouse secondary antibody (1:1000) were consistent with those observed in WB, confirming that CNX may be associated with the occurrence and progression of AD.

[0034] Example 2: Mouse model construction and evaluation

[0035] 1. Model construction:

[0036] The upstream and downstream recombinant homologous fragments of the CNX gene were cloned separately, and the vectors were digested and connected to form the pSYN-CNX-KO knockout vector. Adeno-associated virus was used as the infection vector. 14-week-old mice with normal development were divided into a control group and an experimental group. The control group used an empty virus (AAV-SYN-vecter-GFP), and the experimental group used a CNX knockdown virus (AAV-SYN-CNX-KD-GFP). The effects of the specific knockdown of CNX in adult mice on their cognitive function were evaluated. Adeno-associated virus (AAV-SYN-CNX-KD-GFP) was injected into the hippocampus of mice using a stereotaxic technique. Figure 3 B), behavioral experiments were performed to evaluate the changes in learning and cognitive abilities of neuronal mice 4 weeks after injection ( Figure 3 A).

[0037] After the model was constructed, WB was used to verify the expression of CNX protein in the hippocampus tissue of the two groups of mice ( Figure 3 D and E), the results showed that the expression level of CNX protein in the experimental group was more than 50% lower than that in the control group, indicating that the mouse model was successfully established in the experiment.

[0038] 2. Model Evaluation

[0039] Using the internationally used Morris water maze test mouse model, the mice in the CNX knockdown group took significantly longer to find the platform in the navigation test ( Figure 3 C in the figure), the difference can be further seen from the trajectory diagram ( Figure 3 In the spatial exploration experiment, the distance, time and number of platform crossings in the target quadrant of the CNX knockdown group mice were significantly reduced ( Figure 3H, I, and J in Figure 3). The results of the novel object recognition experiment also showed that the exploration time of CNX knockdown mice was significantly shorter than that of the control mice, and the corresponding cognitive coefficient ( Figure 3 L) and the discrimination coefficient is lower ( Figure 3 The above results showed that the disruption of CNX caused obvious cognitive dysfunction in mice.

[0040] 3. Assessment of Neurons in the Hippocampus After CNX Knockdown

[0041] By TUNEL staining ( Figure 4 The results of A and B in Figure 3 showed that the staining signal intensity of the CNX knockdown group was significantly higher than that of the control group, indicating that the knockdown of CNX exacerbated the cell death in the hippocampus. Figure 3 Figures E and G show that the number of Nissl bodies in the hippocampus of the CNX knockdown group decreased or even disappeared, indicating that the number of active neurons in the mice was significantly reduced. FJC was used to cross-validate the neuronal loss assessed by Nissl staining above. The results showed that the neuronal signal intensity of the CNX knockdown group was significantly higher than that of the control group ( Figure 4 (C and D) indicate that CNX knockdown causes neuronal damage.

[0042] Neurons are important cells in the brain. Information transmission between neurons is achieved through synapses. The main protein markers are postsynaptic density protein 95 (PSD95) and synaptophysin (SYN). Through WB experiments, the levels of neuronal marker protein NeuN and synaptic marker protein PSD95 in the CNX knockdown group were significantly reduced ( Figure 4 These results (F, G, and I) showed that knockdown of CNX in the mouse hippocampus caused significant neuronal damage and loss.

[0043] 4. Evaluation of AD pathological features induced by CNX knockdown

[0044] In addition to obvious neuronal damage, AD is often accompanied by hyperphosphorylation of neuronal microtubules and excessive aggregation of Aβ amyloid protein. Hyperphosphorylation of Tau protein is a hallmark of neurofibrillary tangles (NFTs) in Alzheimer's disease. The phosphorylation levels of Tau protein (T20 and S396) in the hippocampus of CNX knockdown mice were significantly higher than those in the control group ( Figure 5 A, B, C, and D in Figure 3). Specific ELISA was used to detect the expression of Aβ1-40 and Aβ1-42 amyloid proteins. The results showed that after CNX knockdown, the levels of soluble and insoluble Aβ1-42 in the hippocampus were significantly increased ( Figure 5 G and H). All experimental results showed that knockdown of CNX in the hippocampus induced AD-like pathological changes in the hippocampus of mice brain tissue.

[0045] Example Three: Overexpression of CNX in APP mice

[0046] To evaluate the effect of CNX overexpression on cognitive function in adult AD mice, 26-week-old AD mice (APP / PS1) were chosen, and CNX overexpression adeno-associated virus (AAV-SYN-CNX) and empty virus (AAV-vector) were constructed. The AD mice were divided into three groups: blank control group (APP / PS1), empty virus group (APP / PS1-AAV-vector), and overexpression group (APP / PS1-AAV-SYN-CNX). Similarly, the adeno-associated virus of CNX was injected into the hippocampus of AD mice by brain stereotactic technology, and the Morris water maze test and novel object recognition test were performed after 4 weeks.

[0047] First, the CNX overexpression adeno-associated virus (AAV-SYN-CNX) with a neuron-specific promoter SYN and a FLAG tag was used, and the neuron marker (MAP2), astrocyte marker (GFAP), microglia marker (IBA1), and FLAG were co-stained (A in FIG. 6), and it was found that the co-localization of MAP2 and FLAG was obviously more intensive than that of GFAP and IBA1, verifying the specificity of the vector system for neurons. Western blotting was performed on the hippocampal tissues of the three groups of AD mice, and it was found that the CNX protein level in the overexpression group was significantly higher than that in the blank control group and the empty virus group (B and C in FIG. 6); at the same time, it was found that the CNX signal was stronger in the overexpression group by immunofluorescence experiment (D and E in FIG. 6), and the experimental results confirmed that the CNX overexpression system was successfully overexpressed in the hippocampal neurons of AD mice. Figure 6 Figure 6 Next, to evaluate the effect of CNX overexpression on the cognitive function of AD mice, the Morris water maze test was performed in exactly the same way as in Example Two, and the results showed that in the place navigation experiment, the escape time of the CNX overexpression group of mice gradually shortened with the increase of training days, while there was no significant difference in the escape time between the blank control group and the empty virus group (F in FIG. 7), and the same trend could be seen from the trajectory graph (G in FIG. 7). In the spatial exploration test, the distance, time spent, and number of times of crossing the platform in the target quadrant of the CNX overexpression mice were significantly increased (H, I, and J in FIG. 7). The results showed that the spatial memory ability of the CNX overexpression group of mice was improved through learning and training. In the novel object recognition test, the CNX overexpression group of mice showed an increase in the exploration time of the new object compared with the other two groups (M and N in FIG. 7), and the test showed that the cognitive ability of AD mice was partially improved after CNX overexpression. Figure 5

[0048] Figure 6 Figure 6 Figure 6 Figure 6 ​​​​​​​

Claims

1. A method for constructing a mouse model that simulates the natural course of AD, characterized in that: A gene knockout recombinant vector carrying the CNX fragment was introduced into the hippocampus of the brain of adult healthy mice to knock down the CNX gene in the hippocampus, thereby destroying neurons and constructing a mouse model that simulates the natural pathogenesis of AD.

2. The mouse model construction method according to claim 1, characterized in that: CNX gene knockout vector systems include adenovirus, adeno-associated virus, lentivirus, Cre-Lox and CRISPR-Cas systems.

3. The method for constructing a mouse model according to claim 1, comprising the following steps: (1) Clone the upstream and downstream recombinant homologous fragments of the CNX gene respectively, and then digest and ligate them; (2) The cloned fragment in (1) was ligated into the vector to construct the pSYN-CNX-KO knockout vector; (3) Preparation of nucleic acid solution for minimally invasive injection; (4) The constructed recombinant vector is injected into the hippocampus of the brain of adult healthy mice using a minimally invasive injection method, and positive clones are screened.

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