CN118421703B

CN118421703B - Method for constructing non-human mammal model with spontaneous emotion abnormal fluctuation and application thereof

Info

Publication number: CN118421703B
Application number: CN202410603746.5A
Authority: CN
Inventors: 肖潇; 杨智辉; 李明; 王闯; 徐佳; 蔡欣
Original assignee: Kunming Institute of Zoology of CAS; Ningbo University
Current assignee: Kunming Institute of Zoology of CAS; Ningbo University
Priority date: 2024-05-15
Filing date: 2024-05-15
Publication date: 2025-04-29
Anticipated expiration: 2044-05-15
Also published as: CN118421703A

Abstract

The present invention provides a method for constructing a non-human mammal model with spontaneous abnormal mood swings and its use. Specifically, the present invention provides a NEK4 gene expression cassette, a construct containing the NEK4 gene expression cassette, or an expression vector. The present invention also provides a method for constructing an animal model, comprising transferring the NEK4 gene into a non-human mammal brain region or neuron to obtain a non-human mammal model with spontaneous abnormal mood swings that overexpresses the NEK4 gene. The animal model of the present invention is an effective animal model of neuropsychiatric diseases, which can be used to study neuropsychiatric diseases such as bipolar disorder, schizophrenia, depression, anxiety, phobia, autism spectrum disorder, and can be used for screening and testing experiments of specific drugs.

Description

Method for constructing non-human mammal model with spontaneous emotion abnormal fluctuation and application thereof

Technical Field

The invention belongs to the field of biological medicine, and in particular relates to a construction method and application of a non-human mammal model with spontaneous emotion abnormal fluctuation.

Background

Mental diseases refer to a large class of diseases in which mental activities such as cognition, emotion, behavior and the like are damaged to different degrees due to disorder of brain functions, and include severe mental diseases such as schizophrenia, bipolar disorder, depression and the like, and the mental diseases not only seriously affect normal life of patients, but also bring about heavy economic burden to families and society thereof. The current whole genome association study (GWAS) provides a great deal of information about mental diseases, creating the possibility to reveal the pathogenesis of mental diseases. Single Nucleotide Polymorphisms (SNPs) across chromosome 3p21.1 are significantly associated with schizophrenia, bipolar disorder and cognitive function, but their related mechanisms are still rarely reported.

Bipolar disorder is a serious mental disorder affecting more than 1% of the world population and is one of the leading causes of disability worldwide. Mood in bipolar disorder patients has mood swings between mania (or hypomania) and depressed states, which are the most typical symptoms of bipolar disorder. The patient is shown to have increased mood, increased physical activity, reduced sleep, and active thought when in a hypomanic state (at least 4 days), but such a hypomanic state generally does not have a significant impact on normal life. When the patient is in a manic period (at least 7 days), the emotion reaches a peak state, and the patient is extremely active, has improved self-confidence, high emotion, severe reaction, easy impulsivity and irritability, has a sense of everywhere, and also has reduced sleeping time. Mania can also progress to more extreme levels with psychotic-like symptoms, where the patient may develop disorientation (misunderstanding of his own condition or environment), associative impairment (confusion of speech and thinking patterns), and auditory hallucinations, etc. When the patient is in the depressed phase (for 2 weeks), the patient becomes less confident, spelt, low mood, impaired social ability, reduced energy, etc.

Currently, animal models for bipolar disorder are established mainly by controlling genetic risk genes (e.g., SHANK2/3, ANK3, HOMER 1), environmental stimuli, or drug treatments, and abnormalities in the growth, number, and shape of dendritic spines are observed in many of these models, suggesting a potential role for synaptic function in the occurrence of bipolar disorder. However, it is notable that a potential limitation of bipolar disorder studies is that most model animals only exhibit manic, anxiety or depression-like behavior during the course of the experiment, and that it is difficult to simulate the most important clinical signs of bipolar disorder, i.e. the patient's mood can shift between manic (hypomanic) and depressive, which also greatly limits the application of these models in the resolution of pathological mechanisms of bipolar disorder and in the screening of potential drugs.

Thus, there is a strong need in the art to develop a new non-human mammalian model that effectively mimics spontaneous mood swings.

Disclosure of Invention

The invention aims to provide a NEK4 gene expression cassette, a construction or an expression vector containing the NEK4 gene expression cassette and a non-human mammal model for effectively simulating spontaneous abnormal emotion fluctuation. The animal model can simulate a clinical model of abnormal emotion fluctuation of a non-human mammal, and can be widely applied to treatment technology for treating diseases related to abnormal emotion fluctuation and screening and evaluating medicines.

In a first aspect of the invention there is provided the use of a NEK4 gene expression cassette, a NEK4 gene expression cassette-containing construct or an expression vector for the preparation of a formulation for use in constructing a non-human mammalian model for spontaneous occurrence of mood swings.

In another preferred embodiment, the expression vector is a viral vector.

In another preferred embodiment, the viral vector is selected from adenovirus, adeno-associated virus, lentivirus, or a combination thereof.

In another preferred embodiment, the formulation is an intracranial injection formulation.

In another preferred embodiment, the formulation is a gene editing reagent comprising a NEK4 gene expression cassette containing HA homology arms at both ends.

In another preferred embodiment, the gene editing reagent further comprises a Cas9 protein or a nucleic acid encoding the same and a sgRNA.

In another preferred embodiment, the gene editing reagent is for integration of the NEK4 gene expression cassette into the genome of a mouse.

In another preferred embodiment, the integration site is an H11 safe insertion site.

In another preferred embodiment, the NEK4 gene in the non-human mammalian model is overexpressed in brain regions and/or neurons.

In another preferred embodiment, the NEK4 gene is an exogenous NEK4 gene.

In another preferred embodiment, the gene expression cassette comprises a promoter, NEK4 gene and terminator, which are linked in sequence.

In another preferred embodiment, the non-human mammalian model that spontaneously develops mood swings has one or more characteristics selected from the group consisting of:

1) The open field activity level is reduced during the day and/or the open field activity level is normal or elevated at night;

2) The desire to explore new different environments in the daytime is reduced, and/or the desire to explore new different environments in the evening is increased;

3) No or reduced manic behavior during the day, and/or elevated manic behavior at night;

4) The degree of mania decreases during the day and/or the degree of mania increases at night;

5) Increased anxiety-like behavior during the day and/or decreased anxiety-like or anxiety-like behavior during the night;

6) The anxiety level increases during the day and/or the anxiety level decreases during the night.

In another preferred embodiment, the non-human mammalian model is used as an animal model for studying neuropsychiatric disorders including bipolar disorder, schizophrenia, depression, anxiety, phobia, autism spectrum disorder.

In a second aspect of the present invention, there is provided a method for constructing a non-human mammal model in which abnormal emotion fluctuations spontaneously occur, comprising the steps of (a) transferring a NEK4 gene expression cassette, a construct or an expression vector comprising the NEK4 gene expression cassette into a brain region or a neuron of a non-human mammal, thereby obtaining a non-human mammal model in which abnormal emotion fluctuations spontaneously occur, in which NEK4 genes are overexpressed.

In another preferred embodiment, in step (a), the NEK4 gene is transferred into brain regions or neurons of a non-human mammal after viral vector treatment or CRISPR/Cas9 technical condition knock-in treatment, thereby obtaining a non-human mammal model which over-expresses the NEK4 gene and spontaneously generates abnormal emotion fluctuation.

In another preferred embodiment, the method of viral vector treatment comprises the steps of:

(a) Constructing NEK 4-adeno-associated virus expression vectors, and packaging NEK 4-adeno-associated viruses;

(b) Injecting the NEK 4-adeno-associated virus obtained in the step (a) into the dorsal hippocampal brain region of a non-human mammal to obtain an animal model of overexpression of NEK4 genes in the dorsal hippocampal brain region.

In another preferred embodiment, the method further comprises the step (b) of determining the mood swings in the non-human mammalian model by animal behavioural analysis.

In another preferred embodiment, the non-human mammal is a rodent or a non-human primate, preferably comprising a mouse, rat, rabbit, monkey.

In another preferred embodiment, the method of CRISPR/Cas9 technical conditional knock-in treatment comprises:

(1) Introducing sgrnas targeting the H11 site, a targeting vector comprising CAG-LSL-NEK4-HA-polyA and H11 site homologous sequences, and a Cas9 protein into fertilized eggs of a non-human mammal using CRISPR/Cas9 technology to obtain engineered fertilized eggs;

(2) Transplanting the engineered fertilized egg to a female individual of the non-human mammal and producing an F0 generation individual, crossing the F0 generation individual for at least 1 generation to obtain a NEK4- ^flox/flox animal;

(3) And (3) hybridizing the Camk2a-Cre animal with the NEK4- ^flox/flox animal to obtain a non-human mammal model for conditionally over-expressing the NEK4 gene in the forebrain cone neuron.

In another preferred embodiment, the non-human mammalian model is used to screen or identify substances (therapeutic agents) that reduce or treat neuropsychiatric diseases.

In a third aspect of the invention, a non-human mammalian model is provided that spontaneously develops mood swings that overexpress exogenous NEK4 protein in neurons or specific brain regions.

In another preferred embodiment, the exogenous NEK4 protein is selected from the group consisting of a human NEK4 protein, a murine NEK4 protein, or a combination thereof.

In another preferred embodiment, the experimental animal of the non-human mammalian model in which abnormal fluctuations in mood spontaneously occur is prepared by the method according to the second aspect of the present invention or by using the formulation for use according to the first aspect of the present invention.

In a fourth aspect of the invention there is provided a non-human mammalian model prepared by a method according to the second aspect of the invention or the use of a non-human mammalian model according to the third aspect of the invention for screening to determine potential therapeutic agents for the treatment or alleviation of diseases associated with spontaneous abnormal mood swings.

In another preferred embodiment, the screening comprises the steps of administering a candidate substance to the non-human mammalian model to evaluate the effect of the candidate substance on the symptoms of spontaneous mood swings, and optionally, killing the non-human mammalian model.

In a fifth aspect of the invention, there is provided a method of screening or identifying potential therapeutic agents for the treatment or alleviation of diseases associated with spontaneous mood swings comprising the steps of:

(1) In a control group, using the same experimental conditions but applying a solvent that does not contain the candidate substance;

(2) Conduct a behavioral analysis of the behavior of the animal model and compare it to a control group,

Wherein, if the behavior of the disease associated with spontaneous occurrence of abnormal emotion fluctuation is improved in the animal model to which the candidate substance is administered as compared with the control group, it is indicated that the candidate substance can be used as a potential therapeutic agent for the disease associated with spontaneous occurrence of abnormal emotion fluctuation.

In another preferred embodiment, the behavioral analysis is selected from the group consisting of voluntary activity, open field test, elevated plus maze test, forced swimming, or a combination thereof.

In another preferred embodiment, in step (2), when the behavioural analysis indicates a significant improvement in the symptoms of spontaneous occurrence of abnormal mood swings, the candidate substance is suggested to be useful as a potential therapeutic agent for the spontaneous occurrence of abnormal mood swings-related diseases.

In another preferred embodiment, the significance means that there is a statistically significant difference.

It is understood that within the scope of the present invention, the above-described technical features of the present invention and technical features specifically described below (e.g., in the examples) may be combined with each other to constitute new or preferred technical solutions. And are limited to a space, and are not described in detail herein.

Drawings

FIG. 1 shows a graph of the association analysis of the chromosomal 3p21.1 region with the genetic risk of bipolar disorder. Wherein panel A shows that the chromosomal 3p21.1 region contains twenty more genes, where NEK4 is identified as a risk gene for bipolar disorder, and panel B shows that SNPs across the 3p21.1 (hg 19chr3: 52300000-53300000) chromosomal region are analyzed in further detail using GWAS data of PGC3 in relation to risk of bipolar disorder, wherein rs2336147, rs7622851 and rs2276824 each appear to correlate with disease whole genome level (rs 2336147: P=3.62X10 ^-13;rs7622851：P=1.83×10^-11;rs2276824：P=1.02×10^-10).

Fig. 2 shows a graph of the results of analysis of three SNPs (rs 2336147, rs7622851 and rs 2276824) for expression quantitative trait loci (eQTL) using data of hippocampal sample RNA-seq (n=371) in BrainSeq second-stage dataset. Wherein, A represents the eQTL association result diagram of NEK4 gene and rs7622851, rs2336147 and rs 2276824), B represents the eQTL association result diagram of GNL3 gene and rs7622851, rs2336147 and rs 2276824), C represents the eQTL association result diagram of GLYCTK gene and rs7622851, rs2336147 and rs 2276824), wherein GG represents the individual of SNP with G allele on two homologous chromosomes, CG represents the individual of SNP with C allele and G allele on two homologous chromosomes, CC represents the individual of SNP with C allele on two homologous chromosomes, CT represents the individual of SNP with C allele and T allele on two homologous chromosomes, TT represents the individual of SNP with T allele on two homologous chromosomes, and GC represents the individual of SNP with G allele and C allele on two homologous chromosomes.

Figure 3 shows a graph of the results of a biphasic disorder case-control differential expression analysis against a hippocampal dataset. Wherein, the A diagram represents the NEK4 mRNA expression level change in the bipolar disorder case group and the control group, the B diagram represents the GNL3 mRNA expression level change in the bipolar disorder case group and the control group, the C diagram represents the GLYCTK MRNA expression level change in the bipolar disorder case group and the control group, wherein case represents the bipolar disorder case group and control represents the control group.

FIG. 4 shows a schematic representation of NEK 4-adeno-associated virus (AAV) injection protocols and behavioural development protocols. Wherein, the A diagram shows the injection site of the sea horse NEK4-AAV virus on the back side of the mouse and the fluorescent expression schematic diagram, the B diagram shows the time scheme of the behavioural test of NEK4-AAV virus injection mice, wherein, as the ages of the mice increase, NEK4-AAV virus injection (AAV injection) is sequentially carried out on 7-8-week-old mice, open Field (Open Field) experiment is carried out on 11-week-old mice, overhead cross maze (Elevated Plus Maze) experiment is carried out on 12-week-old mice, forced swimming (Forced Swim ming) experiment is carried out on 13-week-old mice, biological rhythm test (Biorhythm detection) is carried out on 15-week-old mice, wherein, +Day1 represents the corresponding behavioural test carried out in daytime on 1 Day of the corresponding week-old, and +Night4 represents the corresponding behavioural test carried out at Night on the corresponding Zhou Lingdi days.

Fig. 5 shows a graph of circadian wave results of adenovirus-associated virus (AAV) -mediated overexpression of NEK4 in the hippocampus leading to mouse emotional behavior in open field experiments. Wherein, the graph A shows the total movement distance (mm) of the mice, the graph B shows the residence time(s) of the mice in the central area of the open field, the graph C shows the residence time(s) of the mice in the corner of the open field, the left side (white background) in the graphs A, B and C shows the result of open field experiments carried out in the daytime, the right side (gray background) shows the result of open field experiments carried out in the evening, the Control shows the Control group, and NEK4 OE shows the NEK4 over-expression group.

FIG. 6 shows a graph of circadian wave results of adenovirus-associated virus (AAV) -mediated overexpression of NEK4 in the hippocampus leading to mouse emotional behavior in an elevated plus maze experiment. Wherein, the graph A shows the total distance (mm) of the mice in all areas of the elevated plus maze, the graph B shows the residence time(s) of the mice in the open arms, the graph C shows the movement distance (mm) of the mice in the open arms, the left side (white background) in the graphs A, B and C shows the results of the elevated plus maze experiment carried out in the daytime, the right side (gray background) shows the results of the elevated plus maze experiment carried out at night, the Control shows the Control group, and NEK4 OE shows the NEK4 over-expression group.

FIG. 7 shows a graph of circadian wave results of adenovirus-associated virus (AAV) -mediated overexpression of NEK4 in the hippocampus leading to emotional behavior of mice in forced swimming experiments, wherein immobility time represents the time of immobility(s) of mice, the left side (white background) represents the results of forced swimming experiments performed during daytime, the right side (gray background) represents the results of forced swimming experiments performed at night, control represents the Control group, and NEK4 OE represents the NEK4 overexpression group.

FIG. 8 shows graphs of the results of free activity of NEK4 overexpressing mice obtained in this example 1 monitored using a small animal metabolic and behavioral phenotyping system for 3 consecutive days. Wherein the left side (white background) in the figure represents the free active path (m) monitored during the day (8 to 20 points), the right side (gray background) represents the free active path (m) monitored at 20 to 7 points at night), control represents the Control group, and NEK4 OE represents the NEK4 overexpression group.

FIG. 9 shows a graph of circadian rhythm fluctuations resulting from NEK4 overexpression causing mouse dorsal hippocampal CA1 dendritic spinogenesis. Wherein, the left side of the A diagram represents a schematic diagram of sparse labeled neurons in the CA1 region of the dorsal hippocampus, red fluorescence indicates injection positions of NEK4 over-expressed AAV, and green fluorescence indicates sparse labeled neurons in the CA1 region. The length of the scale is 100 mu m, the right side of the A diagram is a second-level or third-level branch diagram of the dorsal hippocampus CA1 region sparse marking neuron apical dendrites, the length of the scale is 5 mu m, the Control represents a Control group, the NEK4 OE represents a NEK4 over-expression group, day represents daytime, the Night represents Night, the B diagram represents the influence of NEK4 over-expression on circadian rhythm fluctuation of each subtype of dendrite spines in the neuron apical dendrites, wherein the evaluation criteria of the dendrite spines are as follows, if one dendrite has a head and a neck and the head width is larger than 0.6 mu m, the dendrite spines are classified as Mushroom dendrite spines (Mushroom), and otherwise, the dendrite spines are elongated dendrite spines (Thin). When there is no obvious neck and the aspect ratio is less than 1, the method is classified into a thick short dendritic spine (Stubby), control represents a Control group, NEK4 OE represents a NEK4 over-expression group, control-day and Control-night represent a daytime and evening Control group, NEK4 OE-day and NEK4 OE-night represent a daytime and evening NEK4 over-expression group, respectively, and graph C represents the influence of NEK4 over-expression on the circadian variation of the density of the total dendritic spine of the neuron, wherein the left side (white background) represents the total dendritic spine density measured in daytime, the right side (gray background) represents the total dendritic spine density measured in evening, control represents the Control group, and NEK4 OE represents the NEK4 over-expression group.

FIG. 10 shows a graph of circadian rhythm fluctuations resulting from NEK4 overexpression causing synapse formation in the CA1 region of the dorsal hippocampus of mice. Wherein, panel a represents a photograph taken randomly of dorsal hippocampal CA1 cone neuronal overhead synapses, wherein the upper panel represents a daytime mouse dorsal hippocampal CA1 synapse diagram and the lower panel represents a evening mouse dorsal hippocampal CA1 synapse diagram. Panel B shows a statistical plot of the number of synapses from panel A, with the left side representing the number of synapses during the day, the right side (gray background) representing the number of synapses at night, control representing the Control group, NEK4 OE representing the NEK4 over-expression group. * P is less than or equal to 0.01, P is less than or equal to 0.0001.

Fig. 11 shows a graph of circadian rhythm fluctuations in mouse emotional behavior caused by lithium salt treatment to improve NEK4 overexpression in open field experiments and elevated plus maze experiments. Wherein, graph A shows the total movement distance (mm) of the mice in the open field experiment, graph B shows the distance (mm) of the mice in the open field central area, graph C shows the residence time(s) of the mice in the open field central area, graph D shows the residence time(s) of the mice in the open field corner landing zone, graph E shows the total distance (mm) of the mice in all areas of the elevated cross maze experiment, graph F shows the distance (mm) of the mice entering the open arms in the elevated cross maze, graph G shows the residence time(s) of the mice in the open arms of the elevated cross maze, graph H shows the residence time(s) of the mice in the closed arms of the elevated cross maze, wherein, the left side of each graph shows the corresponding behavior result developed in the daytime, the right side (gray background) shows the corresponding behavior result developed at night, graph Control shows the untreated Control group of lithium salt, graph NEK4 OE shows the overexpressed group of NEK4 treated with lithium salt, graph Control-Li shows the overexpressed group of NEK4 treated with lithium salt. * P is less than or equal to 0.05, P is less than or equal to 0.01.

FIG. 12 shows a graph of the results of lithium treatment to improve the circadian rhythm fluctuations in mouse dendritic spines and synapses caused by NEK4 overexpression. Wherein, graph A shows the effect of NEK4 overexpression in the lithium salt treatment group and the untreated group on circadian variation of density of each subtype of dendrite of pyramidal neurons in the CA1 region of the dorsal hippocampus of the mouse, graph B shows the effect of NEK4 overexpression in the lithium salt treatment group and the untreated group on circadian variation of the number of dendrite formation of pyramidal neurons in the CA1 region of the dorsal hippocampus of the mouse, graph C shows the effect of NEK4 overexpression in the lithium salt treatment group and the untreated group on circadian variation of thickness (PSD thickness) of the compact region after the synapse of the mouse, wherein Control-day represents a Control group which is not treated with lithium salt in daytime, control-day represents a Control group which is not treated with lithium salt in evening, NEK4 OE-day represents a NEK4 overexpression group which is not treated with lithium salt in daytime, control-Li-day represents a Control group which is not treated with lithium salt in evening, and Control-day 4 OE-day represents lithium salt in daytime, NEK-4 OE-day represents lithium-4-over-day. * P is less than or equal to 0.05, P is less than or equal to 0.01, P is less than or equal to 0.0001.

Fig. 13 shows a graph of the effect of lithium salt treatment to improve NEK4 overexpression on GSK3 phosphorylation levels. Wherein, graph A shows GSK3 phosphorylation levels detected by taking Control mice and the back hippocampus of NEK4 over-expressed mice in Day and Night (Night) respectively, two mice in the same group are respectively numbered 1 and 2, such as Control-1 and Control-2 respectively represent the hippocampus of mice numbered 1 and 2 in the Control group, NEK4 OE-1 and NEK4 OE-2 respectively represent the hippocampus of mice numbered 1 and 2 in the NEK4 over-expressed group, graph B shows GSK3 phosphorylation levels detected by taking lithium salt treated and untreated Control mice and the back hippocampus of NEK4 over-expressed mice respectively in Day and Night, two mice in the same group are mixed together as a sample, GAPDH at the lowest position of the graph is a photograph with shorter exposure time, wherein Control represents the untreated Control group of lithium salt, NEK4OE represents the over-expressed group of lithium salt, control-Li represents the Control group of lithium salt treated NEK4 over-expressed group, and Control-Li represents the Control group of lithium salt treated NEK4 over-expressed by lithium salt, and GSK 4P represents GSK3 and GSK3 alpha-expressed by GSK-21 and GSK-3 alpha-GSK-3.

FIG. 14 shows a schematic representation of NEK4 conditional knock-in (NEK 4 cKI) mice propagation strategy and expression validation results. Wherein panel A shows a schematic representation of NEK4 conditional knockin (NEK 4 cKI) mice propagation strategy, wherein NEK4-flox represents male NEK4-flox/flox mice, camk2a-Cre represents female heterozygous Camk2a-Cre mice, NEK4-over expression represents mice specifically knocked in NEK4 genes in the forebrain, especially in CA1 pyramidal neurons of the hippocampus, i.e., NEK4 conditional knockin (NEK 4 cKI) mice, panel B shows a graph of NEK4 expression verification results in NEK4 conditional knockin (NEK 4 cKI) mice, wherein WT-1 and WT-2 represent wild type mice numbered 1 and 2, respectively, NEK4 cKI-1 and NEK4 cKI-2 represent NEK4 conditional knockin mice numbered 1 and 2, respectively.

Fig. 15 shows a graph of circadian wave results of the emotional behavior of NEK4 cKI mice in open field experiments. Wherein, the graph A represents the total movement distance (mm) of the mice in the open field experiment, the graph B represents the distance (mm) of the mice in the open field central area, the graph C represents the residence time(s) of the mice in the open field central area, the graph D represents the residence time(s) of the mice in the open field corner, wherein, the left side (white background) in the graph represents the open field experiment performed in daytime, the right side (gray background) represents the open field experiment performed in evening, the Control represents the Control group mice, and NEK4 ckI represents NEK4 conditional knockout mice.

FIG. 16 shows a graph of circadian wave results of the emotional behavior of NEK4 cKI mice in an elevated plus maze experiment. Wherein, the graph A shows the total movement distance (mm) of the mice in all areas of the elevated plus maze experiment, the graph B shows the distance (mm) of the mice entering the open arms in the elevated plus maze, the graph C shows the residence time(s) of the mice in the open arms of the elevated plus maze, and the graph D shows the residence time(s) of the mice in the closed arms of the elevated plus maze.

FIG. 17 shows a graph of circadian wave results of the emotional behavior of NEK4cKI mice in forced swimming experiments. Wherein, the left side of the graph (white background) represents the forced swimming experiment performed during the day, the right side (gray background) represents the forced swimming experiment performed at night, control represents the Control group mice, and NEK4ckI represents the NEK4 conditional knockout mice.

Detailed Description

Through extensive and intensive studies, the present inventors have established an animal model capable of simulating mood swings in circadian rhythms of patients suffering from bipolar disorder, which can be used as an animal model for studying pathogenesis of bipolar disorder and spontaneous mood swings in screening of new drugs. It is a mouse or other non-human mammal over-expressing the NEK4 gene. The animal model is an effective animal model with spontaneous abnormal emotion fluctuation, can be used for researching neuropsychiatric diseases such as bipolar disorder, schizophrenia, depression, anxiety, phobia, autism spectrum disorder and the like, and can be used for screening and testing experiments of specific drugs.

Terminology

As used herein, the terms "NEK4", "STK2" are used interchangeably and refer to the NEK4 gene or protein component.

As used herein, the terms "model mouse for spontaneous occurrence of mood swings", "model non-human mammal for spontaneous occurrence of mood swings", "animal model" and "model of bipolar disorder" are used interchangeably to refer to the model of non-human mammal for spontaneous occurrence of mood swings as described in the first aspect of the present invention.

NEK4 gene and protein thereof

NEK4 is a member of the family of NIMA kinases (NEKs for short), which in humans contain a total of 11 NIMA kinases, NEK1-11 respectively, all of which possess highly similar kinase domains at the N-terminus, but the regulatory domains at the C-terminus differ significantly in length and composition. This kinase family is another mitotically related kinase family in addition to the Polo and Aurora kinase families, but the cellular biological functions of NIMA kinases are currently less studied, especially in the field of neurobiology. The NIMA kinase family is a serine/threonine kinase, so the earliest name for NEK4 was named kinase-type, called STK2. According to the medium single cell data (https:// www.proteinatlas.org /) of The Human Protein Atlas database, NEK4 was widely expressed in various tissues in humans, with NEK4 being expressed more highly in excitatory neurons, inhibitory neurons, and ciliated cells in addition to spermatogenic cells. Inside the cell, the NEK4 protein is mainly present in the cytoplasm.

NEK4 is present in many different species, and is highly homologous especially in mammals, also being essentially 700-800 amino acids in length (Table A, table B). The human NEK4 gene (ENST 00000233027.10) is located on human genome Chromosome3:52,708,444-52,770,940, the genome is 62597bp in length.

Table A NEK4 genes and proteins

TABLE B homology of partial NEK4 proteins

Studies have shown that the genetic rate of bipolar disorder is higher than 80%. Thanks to the development of the technology, a number of risk gene loci for bipolar disorder are currently identified in the european population by the whole genome association study (GWAS) technique, wherein the chromosome 3p21.1 region is a hot region identified by the GWAS study in which it is identified a number of times as being significantly associated with mental disease, as shown in fig. 1A, which comprises twenty more genes, wherein NEK4 is identified a number of times as a risk gene for bipolar disorder in the GWAS study. Researchers have found that NEK4 is significantly associated with bipolar disorder through a new epigenetic element-based whole transcriptome association study (ETWAS), and in addition, work published in molecular psychiatry by inventor 2019 has also identified NEK4 as an important candidate gene for mental disease using the method of Mendelian randomization (SMR). And indicates that the risk allele of SNPs identified in the 3p21.1 region corresponds to high expression of NEK 4.

The inventors performed a detailed analysis of SNPs spanning the 3p21.1 (hg 19chr3: 52300000-53300000) chromosome region versus risk of bipolar disorder using GWAS data for PGC3, including 41917 clinical cases diagnosed as bipolar disorder and 371549 european blood line control samples. As a result, as shown in FIG. 1B, it was found that 368 SNPs exist in this region and that there are different degrees of Linkage Disequilibrium (LD) with the tag SNPrs2336147 (P=3.62X10 ^-13) located in the gene of PBRM1, and that these 368 SNPs were statistically correlated with the disease relationship to the whole genome level (P.ltoreq.5.00X10 ^–8). In addition, addition of the case samples and control group (including 131969 case samples and 2322416 control samples in total) which self-report as bipolar disorder in PGC4 BD GWAS, the other SNPrs7622851 which has substantially no LD relationship with rs2336147 or rs2276824 (rs 7622851-rs2336147: r ²=0.001;rs7622851-rs2276824:r² =0.001) showed the strongest correlation with bipolar disorder in the 3p21.1 region (p=9.52×10 ^-10), and in PGC3BDGWAS, rs7622851 and rs2276824 also showed correlation with the disease whole genome level (rs 7622851: p=1.83×10 ^-11;rs2276824:P=1.02×10^-10) in addition to rs2336147 (p=3.62×10 ^-13). These data indicate to some extent that there is an independent bipolar disorder risk associated signal for the region 3p21.1 of chromosome.

Subsequently, pheWeb-based whole brain imaging analysis using data from UKbiobank showed that these three independent SNPs (rs 7622851, rs2336147, and rs 2276824) correlated with structural indices of multiple brain regions including the hippocampus.

Thus, the inventors performed analysis of the expressed quantitative trait loci (eQTL) for three SNPs using data of the hippocampal sample RNA-seq (n=371) in the BrainSeq second-stage dataset. As a result, as shown in fig. 2, there were three genes with significant eQTL associations with all three SNPs, including NEK4 (rs 7622851, p=1.01x10 ^-5;rs2336147,P=6.88×10^-11;rs2276824,P=3.79×10^-5, fig. 2A), GNL3 (rs 7622851, p=5.63 x 10 ^-5;rs2336147,P=2.39×10^-10;rs2276824,P=1.56×10^-6, fig. 2B), GLYCTK (rs 7622851, p=2.96 x 10 ^-8;rs2336147,P=1.83×10^-5;rs2276824,P=3.82×10^-5, fig. 2C). At the same time, the risk alleles of the three bipolar disorder risk SNPs are consistent with the high expression of NEK4, GNL3 and GLYCTK MRNA.

Furthermore, in an analysis of the differential expression of the bipolar disorder case-control against the hippocampal dataset, the results are shown in fig. 3, in which case represents the bipolar disorder case group and control represents the control group, fig. 3A shows that NEK4 mRNA expression in the bipolar disorder case group is somewhat elevated compared to the control group (p=0.031, bipolar disorder case group: 7.519 ±0.087, control group: 7.447 ±0.097; mean±s.d), which is consistent with the eQTL analysis results. However, FIG. 3B shows that there was no significant change in the mRNA expression level of GNL3 between the bipolar disorder group and the control group (P=0.834, bipolar disorder case group: 8.918.+ -. 0.068, control group: 8.911.+ -. 0.120; mean.+ -. S.D), and FIG. 3C shows that there was no significant change in the mRNA expression level of GLYCTK between the bipolar disorder group and the control group (P=0.412, bipolar disorder case group: 5.766.+ -. 0.099, control group: 5.800.+ -. 0.146; mean.+ -. S.D). Thus, the present invention is primarily concerned with the NEK4 gene.

Neuropsychiatric disease and disease associated with reduced occurrence of neonatal neurons (neurogenesis)

Neuropsychiatric disorders are a class of disorders of the neural development due to a disorder of the nervous system, or more precisely pathological changes of the cerebral nervous loop, including bipolar disorders, autism spectrum disorders, schizophrenia, depression, anxiety, phobia, epilepsy, etc. The occurrence of serious neuropsychiatric diseases is caused by abnormal development of neurons, improper modification of synaptic connections, and misconnection of nerve loops. Bipolar disorder is a typical neuropsychiatric disorder. Studies have shown that abnormal neuronal growth and abnormal tree emergence lead to bipolar disorder.

Neuropsychiatric diseases and neuronal dendritic spines

Neuronal dendritic spines are generally considered to be sites of excitatory synapse formation, and particularly mushroom dendritic spines possess an enlarged head that can accommodate more postsynaptic receptors and fibronectin for more favorable synaptic structure behavior. Several autopsy brain tissue studies on neuropsychiatric diseases have reported abnormalities in dendritic spines in bipolar disorder, schizophrenia and depression patients, which can be an important internal phenotype of mental disease.

H11 safety site

The random integration transgenic model has the problems of multiple copy number, multiple site insertion, unstable expression, need of line establishment and the like, and the site-directed insertion by using the safe harbor site allows the predictable integration of the exogenous gene into the genome without affecting the activity of the endogenous gene, and the safe harbor site commonly used for the integration of the mouse genome comprises Rosa26, hprt1, H11 and the like. The H11 site (also called Hipp 11) is located on the 11 th chromosome of the mouse and between two commonly expressed and reversely transcribed genes Drg and Eif4enif1, so that the risk of influencing the expression of endogenous genes after insertion of exogenous genes is small, and the site is an open intergenic region without any endogenous promoter element, so that the exogenous genes integrated in the site can be stably expressed under the drive of a designated promoter and support the recombination between chromosomes during meiosis. This gene region has been demonstrated in mice, humans and pigs as a safe harbor site, which is homozygous for the knock-in mouse to develop and reproduce normally. The H11 locus is utilized to construct a conditional knock-in mouse model, so that stable adjustable expression of exogenous genes can be realized, unpredictable abnormal phenotypes possibly caused by over-expression of systemic genes are avoided, and the method is widely used for researching human disease genes. In the project, a CRISPR/Cas9 technology is utilized to insert a CAG-Loxp-Stop-Loxp-cDNA-polyA sequence at an H11 site, namely, a broad-range strong promoter CAG and an exogenous target gene cDNA are separated by a Loxp-Stop-Loxp structure, and only after mating with a tissue-specific Cre tool mouse, the STOP is excised and the target gene can be expressed in specific cells or tissues.

Animal model

The animal model (animalmodelofhumandisease) of human diseases refers to animals with simulated expression of human diseases established in various medical science studies, and spontaneous animal models and induced or experimental animal models are classified by the cause of the generation.

Spontaneous animal model (SpontaneousAnimalModels) refers to a disease that occurs in a natural state in an experimental animal without any conscious artificial treatment. Genetic diseases including mutant lines and tumor disease models of inbred lines. The animal disease model is used for researching the biggest advantage of human diseases, the occurrence and the development of the diseases are very similar to those of human corresponding diseases, the diseases occur under natural conditions, and the animal disease model has higher application value, but the model is difficult to source. Techniques for introducing a target gene by CRISPR-CAS9 technology are known in the art, and these conventional techniques can be used in the present invention.

In the present invention, examples of the non-human mammal include, but are not limited to, mice, rats, rabbits, monkeys, etc., more preferably rats and mice. In the present invention, a fragment containing NEK4 is conditionally knocked into a fertilized egg of a non-mammal to obtain a chimeric mouse containing NEK over-expression, and further, a homozygous mouse containing NEK over-expression is obtained by means of screening, hybridization, and the like. In a preferred embodiment of the invention, mice of homozygous (or heterozygous) type obtained by the invention are both fertile and normally developed. Furthermore, NEK overexpression can be inherited in offspring mice on a Mendelian rule. This shows that the model animal of the present invention is suitable for mass production at low cost.

Construction of animal models

In a preferred embodiment of the invention, NEK4 genes are transferred into a non-human mammal body after being subjected to adeno-associated virus treatment or CRISPR/Cas9 technical condition knock-in treatment, so that an animal model with spontaneous abnormal emotion fluctuation of over-expressed NEK4 genes is obtained.

In a preferred embodiment, the invention provides a NEK 4-adeno-associated virus construction method comprising:

Designing and synthesizing an amplification primer according to the cDNA sequence of the known target gene NEK4, wherein the upstream primer is shown as SEQ ID NO.1, the downstream primer is shown as SEQ ID NO.2, amplifying the target gene NEK4 by a PCR method, and recovering a PCR product by an agarose gel recovery kit after amplification;

Double-enzyme cutting the obtained target gene PCR product by BamHI and HindIII endonucleases, simultaneously cutting the rAAV vector by the two endonucleases, mixing the cut target gene with the rAAV vector, and connecting 1 h, preferably 22 ℃ by using T4 DNA ligase under the condition of 21-23 ℃;

transforming the product obtained by connection into bacterial competent cells, picking up monoclonal and extracting plasmids, transfecting the extracted plasmids into HEK293T cells, culturing for 72 hours, centrifuging the cells, and collecting cell supernatant rich in rAAV;

The cell supernatant was concentrated by purification and the purified NEK 4-adeno-associated virus was collected and stored at-80 ℃.

In another preferred embodiment, purified NEK 4-adeno-associated virus is injected into the dorsal hippocampal brain region of a non-human mammal at a viral titer of 4-5X 10 ¹² GC/mL, preferably 5X 10 ¹² GC/mL, to obtain an animal model for overexpression of NEK4 gene in the dorsal hippocampal brain region.

In another preferred embodiment of the present invention, an animal model for conditional overexpression of NEK4 gene in forebrain cone neurons is obtained by using CRISPR/Cas9 technical conditional knock-in treatment, comprising the steps of:

introducing sgrnas targeting the H11 site, a targeting vector comprising CAG-LSL-NEK4-HA-polyA and H11 site homologous sequences, and a Cas9 protein into fertilized eggs of a non-human mammal using CRISPR/Cas9 technology to obtain engineered fertilized eggs;

transplanting the engineered fertilized egg to a female individual of the non-human mammal and producing an F0 generation individual, crossing the F0 generation individual for at least 1 generation to obtain a NEK4- ^flox/flox animal;

And (3) hybridizing the Camk2a-Cre animal with the NEK4- ^flox/flox animal to obtain a non-human mammal model for conditionally over-expressing the NEK4 gene in the forebrain cone neuron.

Candidate substances or therapeutic agents

In the present invention, there is also provided a method for screening candidate substances or therapeutic agents for treating neuropsychiatric diseases using the animal model of the present invention.

In the present invention, a candidate substance or therapeutic agent refers to a substance known to have a certain pharmacological activity or being detected that may have a certain pharmacological activity, including but not limited to nucleic acids, proteins, carbohydrates, chemically synthesized small or large molecular compounds, cells, and the like. The candidate substance or therapeutic agent may be administered orally, intravenously, intraperitoneally, subcutaneously, intraspinal, or by direct intracerebral injection.

The invention has the following main advantages:

(1) The model mouse for spontaneously generating the abnormal emotion fluctuation can better simulate the abnormal emotion fluctuation spontaneously generated in clinic, in particular to the circadian rhythm emotion fluctuation of a patient with bipolar disorder.

(2) For the spontaneous emotion abnormal fluctuation model mice over-expressing NEK4 genes obtained after CRISPR/Cas9 technical condition knock-in treatment, no difference caused by operation or drug dosage exists among individuals, and the genetic background is highly consistent.

(3) The spontaneous mood abnormality fluctuation model mouse can be used as a powerful tool for researching pathogenesis of bipolar disorder and screening new drugs.

(4) The spontaneous mood abnormality fluctuation model mice of the present invention spontaneously exhibit bipolar disorder-like behavioral symptoms, including behavioral rhythmic changes.

(5) The spontaneous mood abnormality fluctuation model of the invention shows various nerve and mental disease-like symptoms, so the model can be widely used for screening and testing drugs for neuropsychiatric diseases, including bipolar disorder, schizophrenia, depression, anxiety disorder, phobia, autism spectrum disorder and the like.

The invention will be further illustrated with reference to specific examples. It is to be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention. The experimental procedure, which does not address the specific conditions in the examples below, is generally followed by conventional conditions, such as those described in Sambrook et al, molecular cloning, A laboratory Manual (NewYork: coldSpringHarborLaboratoryPress, 1989), or by the manufacturer's recommendations. Percentages and parts are weight percentages and parts unless otherwise indicated.

Unless otherwise indicated, the materials used in the examples were all commercially available products.

Some of the main reagents are summarized in table 1 below:

Table 1:

Example 1 mice obtained with adeno-associated virus (AAV) -mediated overexpression of NEK4 in the dorsal hippocampal brain region

1.1 Construction of NEK4-AAV viral vectors and purification of viral packaging

(1) NEK4 overexpressing plasmid construction AAV overexpressing plasmid pAAV-CAG-tdTomato (# 59262, addgene) the vector was linearized by digestion with BamHI and HindIII enzymes at 37℃for 1h, the digestion system is shown in Table 2 below:

Table 2:

NEK4 (ENST 00000233027) coding sequences were obtained from the Ensembl database, and upstream and downstream primers for NEK4 gene cloning were designed for the coding sequences. As shown in table 3 below:

Table 3:

the cDNA of the cell line is taken as a template, the NEK4 coding sequence is amplified by a PCR method (a PCR reaction system and a program are shown in the table 4 below), whether the size of a PCR band meets the expectations or not is determined by agarose gel electrophoresis, and finally, the NEK4 coding sequence is obtained by cutting, purifying and recycling. The NEK4 PCR product was then digested with BamHI and HindIII enzymes at 37℃for 1h to expose the sticky ends, and the recovered product was purified again for ligation of subsequent expression vectors.

Table 4:

After obtaining the linearized vector and NEK4 coding sequence by the above procedure, the linearized vector and NEK4 PCR product were ligated at 22℃for 1h, the ligation system being shown in Table 5 below. Competent transformation was then performed by adding 50. Mu.L of bacterial competence to the ligation product and placing it on ice for 20min, then immediately placing the competence on ice for 5. 5min after heat shock for 50s in a 42℃water bath, followed by adding 1.1 mLLB to culture and shaking it on a 37℃incubator for 1h to allow the competence to recover activity. After centrifugation at 1000g at room temperature for 10min a of about 900. Mu.L of the medium was removed and the remaining about 900. Mu.L of medium was resuspended in EP tube bottom competence using a pipette. After plating, bacterial monoclonal and grain quality improvement is selected, SANGERDNA sequencing is performed to verify that the NEK4 coding sequence is successfully inserted into the back of the CAG spectral promoter of the pAAV-CAG-tdTomato vector.

Table 5:

(2) Cell plating and transfection healthy HEK293T culture to 90% of the standing dishes for cell passage and cells were plated at a cell density of 4X 10 ⁶ into 150mm dishes for plasmid transfection (cells to about 60%) after 48h of culture. Three plasmid cotransformation (pAAV-CAG-tdTomato or pAAV-CAG-NEK4:10000 ng, AAVDJ:10000 ng, pHelper:20000 ng) was performed in the following proportions, with PEI mediated plasmid transfection into cells, PEI being a cationic polymer with positive charges which, when mixed with plasmid DNA, inter-attract the positive charges of PEI with the negative charges of the plasmid DNA due to electrostatic interactions, forming a polymer complex. PEI encapsulates DNA into positively charged particles that bind to negatively charged cell surface residues and enter the cell by endocytosis of the cell, allowing the expression plasmid to undergo transcriptional translation in the cell.

(3) Cell harvest and NEK4-AAV virus lysis:

After the plasmid was transfected for 72 hours, the cells were scraped and centrifuged at 300 Xg for 10 min at room temperature, and the supernatant was discarded after the completion of centrifugation to obtain cells containing NEK4-AAV viral particles.

After centrifugation, the cells were added with an aqueous lysis buffer (0.15 MNaCl+20mM Tris PH8.0) and resuspended by repeated pipetting with a pipette.

The suspension was placed in liquid nitrogen to quickly form a solid state, which was then thawed in a 37 ℃ water bath. The rapid repeated freeze thawing process was performed 4 times in total to allow the cell membrane to be fully lysed and release NEK4-AAV viral particles inside the cell.

Sodium deoxycholate at a final concentration of 0.5% and Benzonase nuclease at a final concentration of 50U/mL were added to the lysate and incubated in a 37℃water bath for 30 min. Sodium deoxycholate is a water-soluble, cholic acid, anionic detergent, an ionic detergent particularly useful for disrupting and separating protein interactions. Benzonase nuclease can digest nucleic acid released by cell disruption to a length of 3-5 bases completely to avoid the adhesion of lysate.

After the NEK4-AAV virus lysate is incubated, the large cell debris is removed by centrifugation at 10 min at 4 ℃ and 3000 Xg, the supernatant is taken at 4 ℃ and 8000 Xg, and after centrifugation at 60 min, the supernatant is taken to obtain a NEK4-AAV virus crude product (NEK 4-AAV virus containing more impurities).

(4) NEK4-AAV virus super-isolation and purification concentration:

NEK4-AAV virus crude product was added to 15% -25% -40% -54% iodixanol gradient and centrifuged at 350000 Xg at 18℃for 2 hours to ultracentrifuge NEK4-AAV virus crude product.

The 40% iodixanol layer (where NEK4-AAV virus was located after overdose) was extracted with a 10mL injection.

The withdrawn liquid was added to HBSS and centrifuged at 4 ℃,5000×g, 10 min to remove impurities that may be present. The centrifuged virus liquid was transferred to an ultrafiltration tube, washed and purified with HBSS solution, and finally NEK4-AAV virus was concentrated to about 200. Mu.L. NEK4-AAV virus is stored in-80℃refrigerator.

1.2 NEK4-AAV viral titer assay

The pAAV-CAG-tdTomato plasmid was subjected to linearization treatment with KpnI, and the linearized plasmid was purified and recovered after cleavage at 37℃for 1 hour by referring to the above-mentioned 1, NEK4-AAV viral vector construction and viral packaging purification sections.

After the concentration was measured, the sample was set as a standard and diluted at the following concentrations, and the copy numbers were set to 10 ¹⁰、10⁹、10⁸、10⁷、10⁶ and 10 ⁵ vg/mL. mu.L of each purified concentrated NEK4-AAV sample was taken, and the possible plasmid components were removed by treatment with DNaseI (#EN 0529, thermo scientific) at 37℃for 30min, followed by treatment at 95℃for 10 min to inactivate DNaseI, and then NEK4-AAV virus was diluted 1:1000. The Ct values of each gradient standard and NEK4-AAV virus samples were read using the following real-time fluorescent quantitative PCR (RT-qPCR) reaction system and procedure, with the RT-qPCR using ITR specific primers as shown in Table 6 below:

Table 6:

And calculating the virus titer corresponding to the Ct value of the NEK4-AAV virus sample according to the Ct value of each gradient standard substance and the standard curve of known concentration.

1.3 Brain stereotactic injection

7 Week old wild type C57BL/6J mice were used for brain stereotactic injection experiments. The mice were placed in an induction anesthesia box before surgery and were completely anesthetized by an anesthesia machine using isoflurane gas at a concentration of 3% (typically 2-3 min). The isoflurane gas concentration of the anesthesia machine was adjusted to 1% and the gas supply path was switched to the brain location injector mouse mask, after which the head of the fully anesthetized mouse was quickly fixed to the location injector with the ear stem of the location instrument, after which the surgical isoflurane gas concentration was maintained at 1% for continuous anesthesia of the mouse. The hair of the head of the mouse is cleaned by a hair cutter to expose the skin, then the hair of the head of the mouse is wiped with iodine to disinfect the surface, and then the scalp of the mouse is cut off by a small surgical scissors to expose the skull. After the skull is clear and visible, the target brain region is found according to the brain map coordinates of the mouse by taking the bregma as an origin, and a small hole with the diameter of 0.5 mm is drilled on the skull of the mouse by using a cranium drill for delivering NEK4-AAV virus. In this example, NEK4-AAV virus was injected into the dorsal hippocampal brain region of mice, the injection site and fluorescence expression are shown in FIG. 4A, the injection site is 2.06 mm (AP: -2.06 mm) backwards, 1.50 mm (ML: + -1.50 mm) is sideways from the middle suture, and the skull (dura) is downward (DV: -1.50 mm) by bilateral injection. When in injection, the microinjection pump is assembled with a glass tube which is extremely thin and filled with mineral oil and has an extremely thin needle opening and is drawn by a needle drawing instrument, the glass needle is placed above the drilled skull after the microinjection pump sucks NEK4-AAV virus, and the Z-axis knob of the positioning injector is adjusted to enable the glass needle to slowly and slowly descend by 1.5 mm, namely the glass needle reaches the dorsal hippocampus brain region of a mouse. After needle insertion, 3 min was stopped and 200 nLNEK4-AAV was injected at 60: 60 nL/min, with NEK4-AAV titers adjusted to 5X10 ¹² GC/mL. Needle 8-10 min is left after NEK4-AAV virus injection is completed to prevent virus from overflowing to other tissues, and finally the glass needle is taken out. After injection, the skin is sutured and is smeared with sterilizing water for sterilization. The mice after the operation are taken off from the positioning instrument and placed in an electric blanket at 37 ℃ to restore normal consciousness of the mice, and the mice are placed back in a normal feeding environment. For subsequent animal behavioural experiments.

Example 2 behavioural analysis of mice

Significant fluctuations in mood are likely to occur in the face of external stimuli. Light and emotion are one of the very relevant external stimuli, and thus the inventors examined whether daytime and nighttime had an effect on the behavior of NEK4 overexpressed mice obtained in example 1. The following behaviours are developed in the daytime and at night, the time of developing the behavior in the daytime is 9:00 a.m. to 15:00 a.m. (lights on), and the time of developing the behavior in the evening is 21:00 a.m. to 24:00 a.m. (lights off). Meanwhile, in order to avoid the influence on the normal activity state and emotion of the mice when behaviours are developed, each behaviours are separated by more than three days. The time scheme for the behavioral testing of NEK4-AAV virus-injected mice is shown in FIG. 4B.

Mice behavioural development experimental mice were transferred to experimental rooms for one week to adapt to the behavioural room environment. During this period the experimenter gently touched the mice daily to reduce the stress of the mice experimental process. Mice were placed in the behavioural room for 1h in advance before each behavioural test to adapt to the experimental environment. The experimental recording and analysis equipment was purchased from Shanghai Soft information technology Co.

(1) The open field experiment (OpenFieldTest, OFT) is to evaluate the autonomous behavior of the experimental animal in the new and different environment, and the fear of rodents is the new open environment to move mainly in the surrounding environment. The square, non-topped test chamber was equally divided into 16 cells, with the central region 4 cells defined as the central region and each of the 4 corners defined as the corner region (test apparatus 40 cm (length) ×40 cm (width) ×40 cm (height)). The mice were placed in the central area at the time of the experiment, and the time course of the mice in the central area and the total time and course at the four corners within 5 min were tracked and recorded. Mice exhibiting anxiety-like behavior have shorter times or routes to be held in the central region and longer times or routes in the four corner regions.

As a result, as shown in FIG. 5, it was found that there was a significant diurnal variation in the behavior of NEK4 overexpressing mice, and there was no significant difference in the total movement distance between the control group and NEK4 overexpressing group in the open field experiment conducted during the day (control group, 17705.+ -. 2893 mm; NEK4 overexpressing group, 17671.+ -. 6012 mm, df=29, t=0.020, P=0.9842, n=15-16; left side of FIG. 5A). But the time of NEK4 overexpressed mice in the central area of open field was significantly reduced (control, 26.33+ -11.64 s; NEK4 overexpressed, 18.53+ -9.37 s, df=29, t=2.062, P=0.0483, n=15-16; left side of FIG. 5B) and the residence time in the corner of open field was longer (control, 153.0+ -17.79 s; NEK4 overexpressed, 178.3+ -34.49 s, df=29, t=2.542, P=0.0166, n=15-16; left side of FIG. 5C). However, in the open field experiments performed at night, there was no difference in the time for NEK4 overexpressing mice and control mice to enter the central region of the open field (control, 27.80+ -11.85 s; NEK4 overexpressing group, 31.57+ -13.08 s, df=29, t=0.837, P=0.4094, n=15-16; right side of FIG. 5A). These results indicate that NEK4 overexpressing mice transition from daytime anxiety state to nighttime normal state.

(2) Elevated plus maze experiments (Elevated Plus Maze, EPM) the plus maze is a model that uses rodents' exploratory trends in new different environments (open arms) and their own darkness (closed arms) to produce conflicting behaviors, creating anxiety psychology. The overhead plus maze device consists of a closed arm and an open arm crisscrossed with each other from the ground 75 cm. The position where the mice were placed back to the experimenter at the intersection of the open and closed arms was defined as the central area and the mice were facing the open arms, and the time and corresponding distance the mice remained in the open and closed arms within 5 min were tracked and recorded. The higher the anxiety level of the mice, the lower the residence time in the open arm and the distance traveled.

The results are shown in fig. 6, in the elevated plus maze experiments performed during the day, there was no significant difference in total distance between the control mice and the NEK4 overexpressed mice (fig. 6A), but the retention time of the NEK4 overexpressed mice in the open arm (control, 22.38±13.05s; NEK4 overexpressed group, 12.51±9.395 s, df=26, t=2.297, p=0.0299, n=14-14, left side of fig. 6B) and the distance travelled (control, 487.8 ±355.3 mm; NEK4 overexpressed group, 230.1±220.9 mm, df=26, t=2.304, p=0.0294, n=14-14, left side of fig. 6C) were significantly increased compared to the control mice, indicating that the anxiety level of NEK4 overexpressed mice during the day was higher.

However, NEK4 overexpressing mice exhibited the opposite behavior pattern at night, i.e., NEK4 overexpressing mice had a significantly increased residence time on the open arm compared to the control (control, 20.64+ -16.82 s; NEK4 overexpressing group, 44.74 + -20.98 s, df=28, t=3.472, P=0.0017, n=15-15; right side of FIG. 6B), and the distance traveled on the open arm (control, 1011+ -860.0 mm; NEK4 overexpressing group, 1583+ -1008 mm, df=28, t=1.672, P=0.1056, n=15-15; right side of FIG. 6C) had an ascending trend, but did not reach significant levels, indicating that NEK4 overexpressing group mice had elevated exploratory and manic behavior at night.

(3) Forced swimming (Forced Swim Test, FST) the principle of forced swimming is to take advantage of the inherent aversion of rodents to water, which swim surreptitiously in water in an attempt to escape from the water environment. After a period of time, it was found that the animal stopped struggling when no escape was desired, exhibiting a "behavioural desperate" state (floating). Forced swimming is a common method for detecting the antidepressant behavior of experimental mice, so that the forced swimming experiment causes animals to be forced to swim in a limited and non-escape space, and the swimming immobility time is taken as a main index, thereby detecting the despair behavior of the animals. In the experiment, a transparent cylindrical barrel (diameter: 26 cm, height: 40 cm) is filled with water to about 2/3 of the height of the barrel (water temperature is 22-23 ℃), so that feet of a mouse cannot bump on the bottom of the barrel when struggling in water, the immobility time of the 6 min mouse after analysis is continuously recorded for 8 min, and if the immobility time is shorter, the depression degree of the mouse is lower.

As a result, as shown in fig. 7, there was no difference in the immobility time of the NEK4 overexpressed mice compared to the control mice in the forced swimming experiment conducted during the day (control, 134.3±58.36 s; NEK4 overexpressed group, 131.8±59.39 s, df=28, t=0.116, p=0.9086, n=15-15, left side of fig. 7). However, in the forced swimming experiments performed at night, the immobility time of NEK4 overexpressing mice was significantly reduced compared to the control group (control group, 171.6±79.46 s; NEK4 overexpressing group, 109.9±71.63 s, df=28, t=2.234, p=0.0337, n=15-15, right side of fig. 7). This result demonstrates that NEK4 overexpressing mice are more manic at night.

The behavioral results showed that NEK4 can affect the circadian variation of mouse emotion, and dysrhythmia of patients with bipolar disorder is an important condition thereof, so the inventors suspected whether NEK4 affects the biological rhythm of animals. However, when the inventors monitored the free activity results of the NEK4 overexpressed mice obtained in this example 1 for 3 consecutive days using the small animal metabolism and behavior phenotyping system for circadian rhythm disorders, the results are shown in fig. 8, in which NEK4 overexpressed mice have no significant difference in biological rhythm compared to control mice (n=3-3). The results provided by the CircaDB (http:// circadb. Hogeneschlab. Org /) database were also queried, indicating that the mRNA levels of NEK4 were relatively stable between day and night. These results indicate that NEK4 does not affect the biological rhythm of mice.

EXAMPLE 3 dorsal hippocampal neuron sparse labeling and dendritic spine analysis of NEK4 overexpressing mice

The NEK4 is possibly important to control postsynaptic by searching NEK4 interaction protein, and the influence of NEK4 overexpression on excitatory postsynaptic structure, namely neuron dendritic spines, is explored by injecting sparse marker NEK4-AAV virus in a mouse brain stereotactic mode.

(1) NEK4-AAV Virus injection pAAV-CAG-tdTomato or pAAV-CAG-NEK4 over-expressed adeno-associated Virus (AAV) and neuronal sparse marker Virus NCSP-YFP (#BC-SL 001, brainCase) were mixed at 2:1 and the mixed viruses were injected into the dorsal hippocampus of mice as described for 1.3 brain stereotactic injection in example 1. Brain tissue was taken at day and night around 3 weeks of injection to observe the effect of NEK4 overexpression on mouse neuronal dendritic spines, respectively.

(2) Heart infusion in mice were weighed and ketamine was injected intraperitoneally with a syringe based on the mice weight (about 10 g mice were injected with 0.06 mL, adult mice were typically injected with 150 μl). After the mice were fully anesthetized, the mice were fixed with their abdomen facing upward on a foam plate, and then the whole heart was exposed using surgical scissors to shear the mouse chest and ribs. The needle for perfusion is inserted into the left atrium from the apex of the heart and the right auricle is cut off, then the physiological saline is used for perfusion and evacuation of blood in the body until the liver is obviously whitened, finally the heart perfusion is carried out again by using 4% paraformaldehyde to pre-fix the tissues, the mouse brain is taken out through dissection and the mouse brain is soaked by using 4% paraformaldehyde in the 4 ℃ environment overnight for re-fixing.

(3) And (3) cerebral tissue dehydration, namely placing the rat brain subjected to fixation treatment in a side swing table for three times with PBS, and carrying out gradient dehydration on the cerebral tissue after each time for half an hour. The brain tissue was first immersed in 8 mL volumes of 20% sucrose in PBS, placed in a 4 ℃ refrigerator until the brain tissue settled to the bottom of the tube, and then replaced with 30% sucrose in PBS for related operations.

(4) And (3) embedding and slicing brain tissues, namely taking out the samples when the brain tissues are immersed into a PBS solution containing 30% of sucrose to the bottom of the tube, sucking the samples clean by using a residual sucrose solution on the surfaces of the water-absorbing paper brain tissue samples, putting the samples into a square embedding mould, adding an embedding agent OCT into the mould, and quick-freezing and embedding the samples in a refrigerator at-80 ℃. The embedded samples were equilibrated in a microtome for about 1h before being sectioned and then the brain tissue samples were cut into 50 μm thick slices using a frozen microtome, and the brain slices were harvested from the dorsal hippocampal brain area and placed in 24-well plates with PBS.

(5) Immunofluorescent staining of brain pieces the cut brain pieces were washed three times with PBS to remove as much as possible the embedding medium OCT, and then the brain pieces were blocked with a PBS solution of 2% goat serum for one hour at room temperature. The blocking solution was then removed, and the primary antibody was incubated overnight at 4 ℃ in a refrigerator with 2% goat serum +0.2% tritonx-100+ anti flag antibody PBS solution. The next day after removal of primary antibody liquid, the samples were washed three times with PBS, once every 10 min. The secondary antibody was then incubated at room temperature for 1 hour with the addition of 2% goat serum +0.2% tritonx-100+ alexafluor594 antibody PBS solution. Finally, the cells were stained with nuclei (1:1000 dilution in PBS) after incubation at room temperature of 10 minDAPI, and then the cells were blocked after three more washes with PBS.

(6) And (3) shooting and analyzing the nerve cell dendritic spines, namely picking and shooting the 2-3 branch dendrites of the dorsal hippocampus CA1 vertebral nerve cell apices. The dorsal hippocampal CA1 neurons were randomly photographed using a Zeiss confocal microscope (LSM 880 Basic Operation) layer-scan (Z-Stack) system (41 pictures were continuously taken at a resolution of 1024X 1024 pixels, 0.25 μm step pitch using a 100-fold mirror). In each batch of experiments, about 20 neurons were randomly photographed from about 8 brain slices and the experiment was repeated 3 times. NeuronStudio software was used to analyze the shape and density of dendritic spines secondary or tertiary to neuronal dendrites in the captured pictures. Each neuron analyzes at least more than two branches and then averages the resulting values as the respective types of dendritic spine densities and total dendritic spine densities for that neuron. The criteria for the dendritic spine subtypes are classified as mushroom-shaped dendritic spine (mushroom) if one dendritic spine has a head and neck and a head width greater than 0.6 μm, and as elongated dendritic spine (thin). When the dendritic spine has no obvious neck and the aspect ratio is <1, the dendritic spine is classified as thick and short (stubby). The difference in dendritic spines between experimental groups was assessed using a two-tailed t-test and Bonferronipost-hoc two-factor anova. * P is less than or equal to 0.05, P is less than or equal to 0.01, P is less than or equal to 0.005, P <0.0001.

The results are shown in fig. 9A, but NEK4 overexpression resulted in circadian fluctuations in dendritic spine morphology. Specifically, no difference was found between NEK4 overexpressing mice and control group of short dendritic spines (left Stubby of fig. 9B) and total dendritic spines density (fig. 9C), whether day or night.

However, as shown in the Mushroom group on the right side of fig. 9B, NEK4 overexpressed mice had higher Mushroom-like dendritic spine density during the day than the control group (control group, 3.885 ±0.645; NEK4 overexpressed group, 5.366±0.973, df=99, t=3.541, p=0.0018, n=19-16), whereas at night, the trend was reversed, i.e., NEK4 overexpressed mice had lower Mushroom-like dendritic spine density than the control group (control group, 5.196±0.860; NEK4 overexpressed group, 3.243 ±0.823, df=75, t=3.815, p=0.0008, n=14-13), indicating that NEK4 had a strong effect on dendritic spine in a circadian fashion.

Furthermore, as shown in the middle Thin group in fig. 9B, NEK4 overexpression resulted in a significant decrease in the density of elongated dendritic spines during daytime (control group, 11.587 ±1.867; NEK4 overexpression group, 9.220 ±2.054, df=99, t=5.663, p <0.0001, n=19-16), while this effect disappeared at night.

Notably, the above results also show that the mouse mushroom-shaped dendritic spines were more dense in the evening (Control-night) than in the daytime (Control-day) and less elongated dendritic spines (daytime: mushroom-shaped dendritic spines, 3.885 ± 0.6455; evening: mushroom-shaped dendritic spines, 5.196±0.8599, df=31, t=5.009, p <0.0001, n=19-14. Daytime: elongated dendritic spines, 11.59 ±1.867; evening: elongated dendritic spines, 10.25±2.180, df=31, t=1.895, p=0.0675, n=19-14), which may be relevant to the mouse being a viable animal.

Neuronal dendritic spines are generally considered to be sites of excitatory synapse formation, and particularly mushroom dendritic spines possess an enlarged head that can accommodate more postsynaptic receptors and fibronectin for more favorable synaptic structure behavior. It was then further investigated whether overexpression of NEK4 in mice would cause a change in synaptic structure. Transmission electron microscopy image analysis was performed on hippocampal tissue of mice injected with NEK4 overexpressing virus in stereotactic (double-blind random collection of the apical dendrite position of hippocampal CA1, area 19 μm ², magnification 20000, as shown in FIG. 10A).

As shown in fig. 10B, the number of synapses formed per unit area of the hippocampal CA1 region in NEK4 overexpressed mice and control mice also differed significantly, and also had circadian rhythmicity. NEK4 overexpressing mice had significantly more synapses during day time than the control group (control group, 8.421 ±2.143; NEK4 overexpressing group, 11.79 ±2.070, df=36, t=4.928, p <0.0001, n=19-19), but NEK4 overexpressing mice had significantly lower synapses during night time than the control group (control group, 8.421 ±2.143; NEK4 overexpressing group, 11.79 ±2.070, df=37, t=3.481, p=0.0013, n=19-19). Thus, the results of both dendritic spines and synaptic structures demonstrate that NEK4 has an effect on the circadian rhythm of synapse formation.

Example 4 lithium salt improves circadian fluctuations in mouse emotional behavior caused by NEK4 overexpression

Bipolar disorder currently does not have a gold standard to measure that an animal model is bipolar. The above example observes that overexpression of NEK4 in the hippocampus of mice causes circadian fluctuations in the emotional behavior of mice, and in order to better illustrate the relationship between NEK4 gene and the onset of bipolar disorder, the present example further uses clinical drugs to explore whether drugs can improve the emotional disorder of mice caused by NEK4 overexpression. Mood instability is a critical clinical manifestation in patients with bipolar disorder and lithium salts are the most common mood stabilizer in treatment of bipolar disorder, so the effect of lithium salts on the mood behavior of NEK4 overexpressing mice was explored. Meanwhile, in order to simulate the blood lithium concentration of clinical treatment, the inventor adopts intraperitoneal injection of lithium salt to rapidly increase the blood lithium concentration of the mice, and then the mode of adding lithium salt into drinking water of the mice is adopted to maintain the blood lithium concentration of the mice at a certain concentration as a result of drug treatment. Lithium salts have a relatively slow onset in the clinic and are therefore also administered by chronic treatment. The behavioural test was performed on experimental mice after two weeks of drug treatment. The specific behavioural procedure is described with reference to example 3, the results are shown in figure 11:

(1) For open field experiments, in the lithium salt untreated mice, NEK4 overexpressed mice were still observed to have higher anxiety levels during the day compared to control mice (NEK 4 overexpressed mice had less total distance (fig. 11A) and less distance into the central area (fig. 11B), and longer residence time in the corner areas (fig. 11D), but NEK4 overexpressed mice had comparable emotional behavior to control mice at night. Whereas in the lithium salt treated mice, lithium salt significantly reduced daytime anxiety-like behavior in the NEK4 overexpressed mice, i.e., the lithium salt treated NEK4 overexpressed mice had longer residence time into the open field central region (fig. 11C) compared to the untreated mice, less residence time in the open field corner region (fig. 11D), and no significant difference compared to the wild type mice.

(2) For the elevated plus maze experiment, in the lithium salt untreated mice, the NEK4 overexpressed mice were also observed to have higher anxiety levels during the day (overall NEK4 overexpressed mice distance (fig. 11E) and less distance and residence time into the open arms (fig. 11F) and longer residence time in the closed arms (fig. 11H) than the control mice), but significantly lower anxiety levels during the night (NEK 4 overexpressed mice distance and residence time into the open arms (fig. 11F) and shorter residence time in the closed arms (fig. 11H) than the control mice). Whereas in the lithium salt treated group, lithium salt significantly reduced daytime anxiety-like behavior and nighttime mania-like behavior in NEK4 overexpressed mice. Taken together, NEK4 overexpressing mice exhibit fluctuations in mood-related behavior that can be saved by lithium salt treatment, suggesting that these mice may mimic abnormal mood transitions in patients with bipolar disorder.

Example 5 lithium salt improved circadian fluctuation of mouse dendritic spines and synapses caused by NEK4 overexpression

After observing that lithium salt improves the circadian fluctuation of mouse emotion behaviors caused by NEK4 overexpression, the influence of lithium salt treatment on the synaptic structure of NEK4 overexpressed mice is further explored. The results are shown in fig. 12, wherein,

FIG. 12A shows that NEK4 overexpression in the lithium salt untreated group alters morphology of day and night dendritic spines, especially in mushroom dendritic spines (Control-day group: 3.354.+ -. 0.4261; NEK4 OE-day group, 4.553.+ -. 0.9704, df=69, t=3.471, P=0.0027, n=12-13.control-day group, 4.684.+ -. 0.5967; NEK4 OE-day group, 2.463.+ -. 0.8469, df=72, t=5.252, P <0.0001, n=13-13). However, lithium salt treatment reduced diurnal variability in abnormal morphology of NEK4 overexpressed mouse dendritic spines (especially mushroom dendritic spines) (Control-Li-day group: 3.557 + -0.2477; NEK4 OE-Li-day group: 3.144 + -0.6154, df=69, t=1.043, P=0.6058, n=12-13.control-Li-day group: 4.392 + -0.6069; NEK4 OE-Li-day group: 4.295+ -0.6461, df=75, t=0.3374, P=0.9818, n=13-14).

FIG. 12B also shows that NEK4 overexpression causes abnormal numbers of synapses in day and night in the lithium salt untreated group (Control-day group: 9.893.+ -. 2.149; NEK4 OE-day group: 11.760.+ -. 2.444, df=55, t=3.056, P=0.0035, n=28-29. Control-day group: 11.570.+ -. 2.373; NEK4 OE-day group: 9.214.+ -. 3.281, df=56, t=3.144, P=0.0027, n=30-28). While lithium salt treatment reduced the diurnal aberrant change in synaptic numbers caused by NEK4 overexpression (Control-Li-day group: 10.90.+ -. 2.119; daytime: NEK4 OE-Li-day group: 11.18.+ -. 2.653. Control-Li-light group: 11.57.+ -. 2.348; NEK4 OE-Li-light group: 11.34.+ -. 2.497).

To further explore the effect of NEK4 on the structure and function of synapses, the inventors have studied the structure of the above mouse postsynaptic compact region (PSD) at the same time. Fig. 12C shows that in the lithium salt untreated group, the control mice had slightly increased PSD thickness at night compared to daytime (daytime: 26.71± 8.263; evening: 31.81±4.713, df=43, t=2.604, p=0.0126, n=20-25). Meanwhile, NEK4 over-expressed mice were found to have increased PSD thickness during the day and decreased PSD thickness during the night compared to the control group (day: control group, 26.71.+ -. 8.263; day: NEK4 over-expressed group, 33.58.+ -. 9.178, df=38, t=2.478, P=0.0173, n=20-20. Evening: control group, 31.81.+ -. 4.713; evening: NEK4 over-expressed group, 23.99.+ -. 6.117, df=45, t=4.940, P <0.0001, n=25-22). Meanwhile, it was consistently found that lithium salt treatment reduced abnormal changes in PSD thickness caused by NEK4 overexpression (during the day: control-Li group, 28.59.+ -. 5.414; NEK 4OE-Li group: 26.45.+ -. 4.810. During the night: control-Li group, 33.43.+ -. 6.183; NEK 4OE-Li group: 35.51.+ -. 8.283).

Example 6 NEK4 overexpression Down-regulates lithium salt target GSK3 beta phosphorylation but does not exhibit circadian fluctuation

Bipolar disorders currently rely primarily on drug therapy clinically, including mood stabilizer lithium salts, are commonly used in clinical therapies. Although lithium salts have been used as first-line drugs for the treatment of bipolar disorders for a relatively long time, their use in the treatment is not medically designed drugs, but rather, it has been found that lithium salts have therapeutic effects on psychotic bipolar disorders in the event of a symptomatic coincidence, and in fact, therapeutic drugs for many psychotic diseases are inadvertently found, and the mechanism of action of many psychotic drugs including lithium salts is not well defined. Lithium salts are now considered to be multi-targeted, but it is now clear that lithium salts (KleinandMelton, 1996) are inhibitors of GSK-3β. Studies of multiple clinical samples have found that the phosphorylation levels of GSK-3 beta are altered, and in particular that both sites S9 and S21 show reduced phosphate levels in samples derived from bipolar disorder patients. The main mechanism of GSK3 activity regulation is phosphorylation of its N-terminal serine (S) residue, wherein GSK3 beta is mainly related to the degree of S9 phosphorylation, GSK3 alpha is mainly related to the degree of S21 phosphorylation, and phosphorylation at these two sites has an inhibitory effect on GSK3 kinase activity. GSK-3β overactivity is currently thought to be one of the causes of bipolar disorder, whereas lithium salts can inhibit GSK-3β overactivity.

In this example, the effect of NEK4 overexpression on GSK3 beta phosphorylation levels in hippocampal tissue of mice in the lithium salt treated group and the untreated group was examined by western blot. As a result, as shown in fig. 13, NEK4 overexpression significantly reduced the phosphorylation levels of the corresponding S21 and S9 sites of gsk3α and GSK-3β, both day and night (fig. 13A and 13B). And the lithium salt treatment can recover the change of the low phosphorylation to a certain degree.

Example 7 NEK4 conditional knockin mice resulted in circadian fluctuations in emotional behavior

7.1 Obtaining mice specifically knocked in NEK4 Gene in hippocampal CA1 Cone neurons and forebrain

Wild type C57BL/6J mice were purchased from Jiangsu Jiugang Biotech Co.

(1) Knock-in Gene the relevant information is shown in Table 7:

Table 7:

(2) And (3) constructing a carrier:

According to the design scheme, gRNA (CTGAGCCAACAGTGGTAGTA) is designed, constructed and transcribed in vitro, and a homologous recombinant vector (targeting vector: homologous arm containing CAG promoter-Loxp-Stop-Loxp-NEK 4-HA-WPRE-polyA and H11 site) is constructed simultaneously to target on a safety site H11 common to a gene knock-in mouse, and the correctness of the vector sequence is verified by sequencing. The relevant primer sequences are shown in Table 8 below:

Table 8:

(3) Microinjection and identification of F0 mice:

Microinjection of Cas9, gRNA and targeting vector samples into fertilized eggs of mice with C57BL/6JGpt background, transplanting the fertilized eggs which survive the injection into pseudopregnant female mice, and carrying out pregnancy on the mice. F0 generation young animals born by the recipient mice are cut off the tail and toe numbers in 5-7 days, and genome DNA is extracted for PCR and sequencing identification, so that the genotype is confirmed. The F0 generation mouse obtained by the fertilized egg injection method is possibly chimeric/heterozygous/homozygous, and the genotype of the F0 generation mouse obtained by carrying out genotype identification on the tail of the F0 generation mouse is only used as a reference, and cannot represent that the F0 generation mouse is necessarily heritable gene mutant type, and the heritable genotype is required to be determined after the genotype identification of the F1 generation mouse.

(4) Breeding positive F0 mice:

Mating the positive F0 generation mice with wild background mice after sexual maturity, cutting tail and cutting toe numbers of the born F1 generation mice in 5-7 days, extracting genome DNA, carrying out PCR and sequencing identification, and confirming genotype. The relevant primer sequences are shown in Table 9 below:

Table 9:

(5) Propagation of F1 mice and mating with Camk2a-Cre tool mice:

The gene expression of NEK4-HA is in a closed state before the model is matched with Cre tool mice. After mating with Cre tool mice, NEK4-HA gene can be overexpressed in specific tissues or cells. First, flox male mice homozygous for NEK4cKI were obtained by genotyping, and hybridized with Camk2a-Cre (JAX: 005359) positive tool female mice (avoiding germline leakage) to obtain animal models of NEK4 gene specific overexpression in forebrain, especially hippocampal CA1 pyramidal neurons (FIG. 14A). Overexpression of NEK4 was verified by Western blot (FIG. 14B) for subsequent experiments.

NEK4- ^flox/flox mice and Camk2a-Cre tool mice were housed in a clear, independently ventilated standard cage (IVC) in 4-5 mice cages. The mice were free to gain food and water, the feeding room for the mice was set to a 12 hour light/dark cycle (on at 08:00 and off at 20:00, respectively) and the temperature was set to 22 ℃. All experiments were approved by the animal ethics committee of the Kunming animal research institute (NO: SMKX-2021-11-001), in compliance with the national institutional council guidelines for laboratory animal research.

7.2 Behavioural analysis of NEK4 conditional knock-in mice

The detailed results of the behavioural test in this example are as follows with reference to the method described in example 2:

(1) In open field experiments, the results are shown in fig. 15, with NEK4 conditional knockout mice (NEK 4 ckI) transitioning from a daytime anxiety state to a normal night state. Specifically, NEK4 conditional knockout mice did not significantly differ from control mice in total range of motion (fig. 15A) and in corner area residence time (fig. 15D), but NEK4 conditional knockout mice had less day time to central area distance (fig. 15B) than control mice (control, 3132±1266mm; NEK4 cKI group, 1862±946.4mm, df=14, t=2.272, p=0.0393, n=8-8), but at night there was no difference between the two groups of mice. At the same time, the same phenomenon was also observed in the central zone residence time (fig. 15C).

(2) In the overhead plus maze, as shown in fig. 16, the total distance traveled by the NEK4 cKI mice during the day (fig. 16A) and the distance traveled in the open arm (fig. 16B) were significantly higher than those of the control mice, and the residence time of the NEK4 cKI mice during the day into the open arm (fig. 16C) was significantly less than those of the control mice, and the residence time in the closed arm (fig. 16D) was significantly more than those of the control mice (open arm time: control group, 55.83±26.52 s; NEK4 cKI group, 17.10±10.64 s, df=14, t=3.833, p=0.0018, n=8-8. Closed arm time: control, 205.0±4 cKI group, 261.3±13.74 s, df=14, t=4.363, p=0.0006, n=8-8). While at night they exhibited the opposite behavior pattern, but the NEK4 cKI mice had significantly more residence time on the open arm (FIG. 16C) than the control group and significantly less residence time on the closed arm (FIG. 16D) than the control group (open arm time: control group, 28.77.+ -. 12.84 s; NEK4 cKI group, 50.87.+ -. 17.89 s, df=14, t=2.839, P=0.0131, n=8-8. Closed arm time: control group, 256.1.+ -. 19.93 s; NEK4 cKI group, 231.4.+ -. 19.03 s, df=14, t=2.535, P=0.0238, n=8-8).

(3) In the forced swimming experiment, the movement state in the mouse 8 min is detected, and the stationary time of the 6 min mice is kept after analysis. As a result, as shown in fig. 17, depression-like moods of NEK4 cKI mice were observed to be different in the daytime and at night, although statistical significance was not achieved on experimental data. Taken together, these results indicate that NEK4 cKI mice cause circadian rhythms of emotional behavior to fluctuate.

Discussion of the invention

Mood swings are the most typical clinical feature of bipolar disorders, but the biological mechanisms behind mood swings are still poorly understood. Several animal models have been successfully constructed so far that can mimic mania-like behavior, including regulation of circadian genes, such as Clock-19 mutant mice (royballetal, 2007), sleep deprivation (gessa et al, 1995), and administration of cocaine, amphetamines and psychostimulants including amphetamine dialkyl esters, etc., which make a prominent contribution to understanding bipolar disorder (Mac e doet al, 2013). However, few animal models exhibit spontaneous mood changes similar to those of patients with bipolar disorder.

NEK4 is located in the 3p21.1 chromosomal region, one of the most important genetic loci in the bipolar disorder GWAS study, and the inventors demonstrated that independent bipolar disorder risk SNPs in this region consistently predicted higher NEK4 mRNA expression levels in the hippocampus. And early studies showed that overexpression of this gene resulted in altered morphology of primary cultured neuron dendritic spines.

The inventor constructs NEK4 over-expressed mice by means of transgene and AAV virus injection for the first time, truly simulates spontaneous fluctuation between daytime anxiety-like behaviors and nighttime mania-like behaviors found clinically for the first time, and is hopeful to build an animal model with wide application value and spontaneous occurrence of abnormal emotion fluctuation.

The invention explores the mechanism of emotion transition through this animal model. Although NEK4 overexpressed mice exhibit circadian rhythm fluctuations in mood-related behaviors, the gene does not affect the circadian biological rhythms of the mice, suggesting that it may affect biological events that follow circadian rhythm changes.

Second, the inventors observed that the mice' active status and their synaptic structural changes were consistent in control mice, and all of these indicators were significantly elevated during the night. While this finding may reflect the activity status of the mouse nocturnal animal, it also emphasizes the presence of steady-state fluctuations in synaptic structure in a circadian fashion. This form of presence of synapses has also been recently discovered, greatly facilitating understanding of synaptic function (devivotetal, 2017). The present invention found that NEK4 overexpression resulted in a significant disruption of this synaptic homeostasis dynamics, i.e., NEK4 overexpression resulted in an increase in mushroom dendritic spine density, synaptic number and PSD thickness during the day, and a decrease at night, accompanied by the development of behavioral abnormalities. Thus, it is speculated that a disturbance of the synaptic "internal clock" may explain the effect of this gene on emotional behavior.

Furthermore, the inventors further found that lithium salt treatment restored synaptic homeostasis while also improving the behavioral abnormalities in NEK4 overexpressing mice. Notably, lithium salt treatment also resulted in a slight trend of slightly lower daytime activity and slightly higher nighttime activity in control mice, which may reflect the findings that previous studies indicated that lithium salts could enhance circadian amplitude (MCCARTHYANDWELSH, 2012).

Through comprehensive evaluation, the animal model of the invention mainly reflects the disease characteristics of bipolar disorder and can be used as an animal model for screening medicines for treating bipolar disorder diseases. Meanwhile, the method is expected to be used for researching the action relation between NEK4 and other pathogenic genes of the bipolar disorder disease so as to further analyze the pathogenesis of the bipolar disorder disease.

The inventor utilizes the advanced CRISPR-Cas9 assisted transgenic technology and adeno-associated virus transfection technology to construct a non-human mammal model with spontaneous abnormal emotion fluctuation for the first time. NEK4 is over-expressed in this model. The disease model of the invention can be used for carrying out functional analysis on the organism level of NEK4 so as to determine the etiology and pathogenesis of NEK4 in the bipolar disorder disease, and has important scientific significance. In addition, by carrying out drug treatment on the animal model, the method is expected to lay a foundation for developing new control technology and drugs.

All documents mentioned in this disclosure are incorporated by reference in this disclosure as if each were individually incorporated by reference. Further, it will be appreciated that various changes and modifications may be made by those skilled in the art after reading the above teachings, and such equivalents are intended to fall within the scope of the application as defined in the appended claims.

Claims

1. The use of NEK4 gene expression cassette, NEK4 gene expression cassette-containing construct or expression vector for the preparation of a formulation for the construction of a non-human mammalian model in which abnormal fluctuations in mood spontaneously occur, NEK4 gene overexpression in brain regions and/or neurons, said non-human mammalian model exhibiting bipolar disorder-like behavioral symptoms by itself,

Wherein the mood swings include circadian mood swings, wherein the NEK4 gene is a human NEK4 gene.

2. The use according to claim 1, wherein the expression vector is a viral vector.

3. The use according to claim 2, wherein the viral vector is selected from adenovirus, adeno-associated virus, lentivirus, or a combination thereof.

4. The use according to claim 1, wherein the formulation is an intracranial injection formulation.

5. The use according to claim 1, wherein the preparation is a gene editing reagent comprising a NEK4 gene expression cassette containing HA homology arms at both ends.

6. The use according to claim 5, wherein the gene editing reagent is for integration of the NEK4 gene expression cassette into the genome of a mouse.

7. The use according to claim 6, wherein the site of integration is an H11 safety insertion site.

8. The use according to claim 1, wherein in said non-human mammalian model, NEK4 gene overexpression leads to circadian fluctuations in dendritic spinogenesis.

9. The use according to claim 1, wherein said non-human mammalian model of spontaneous mood swings, as compared to a wild type control animal, has one or more characteristics selected from the group consisting of:

1) The activity level of the open field is reduced in the daytime, and the activity level of the open field is normal or increased at night;

2) The desire to explore new different environments in the daytime is reduced, and the desire to explore new different environments in the evening is increased;

3) No or reduced manic behavior during the day, and elevated manic behavior at night;

4) The mania degree is reduced in the daytime and the mania degree is increased at night;

5) Increased anxiety-like behavior during the day, and no anxiety-like behavior or decreased anxiety-like behavior during the night;

6) The anxiety level increases during the day and decreases during the night.

10. The use according to claim 1, wherein the non-human mammalian model is an animal model for studying neuropsychiatric disorders including bipolar disorder, schizophrenia, depression, anxiety, phobia, autism spectrum disorders.

11. A method for constructing a model of a non-human mammal in which abnormal fluctuations in emotion occur spontaneously, comprising the steps of:

(a) Transferring the NEK4 gene expression cassette, a construction or an expression vector containing the NEK4 gene expression cassette into brain regions or neurons of a non-human mammal, thereby obtaining a non-human mammal model with spontaneous abnormal emotion fluctuation of over-expressed NEK4 genes, wherein the NEK4 genes are human NEK4 genes.

12. The method of claim 11, wherein in step (a), the NEK4 gene is transferred into brain regions or neurons of a non-human mammal after viral vector treatment or CRISPR/Cas9 technical conditional knock-in treatment to obtain a non-human mammal model in which abnormal mood swings spontaneously occur over-expressing the NEK4 gene.

13. The method of construction according to claim 12, wherein the method of viral vector treatment comprises the steps of:

14. The method of claim 11, further comprising the step of (b) determining the mood swings of the non-human mammalian model by animal behavioural analysis.

15. The method of claim 11, wherein the non-human mammal is a rodent or a non-human primate.

16. The construction method according to claim 12, wherein the CRISPR/Cas9 technical conditional knock-in treatment method comprises:

17. The method of claim 16, wherein the non-human mammalian model is used to screen or identify therapeutic agents that reduce or treat neuropsychiatric disorders.

18. The method of claim 11, wherein the non-human mammal comprises a mouse, a rat, a rabbit, or a monkey.

19. Use of the non-human mammalian model prepared by the construction method of claim 11, for screening to determine potential therapeutic agents for treating or alleviating a disease associated with spontaneous mood swings.

20. A method of screening or identifying potential therapeutic agents capable of treating or alleviating a disease associated with spontaneous mood swings comprising the steps of:

(1) In the control group, the same experimental conditions are adopted but the applied substances are replaced by corresponding solvents which do not contain the candidate substances;

(2) Performing a behavioral analysis of the behavior of the animal model of (1) and comparing it to a control group;

21. The method of claim 20, wherein the behavioral analysis is selected from the group consisting of autonomous activity, open field testing, elevated plus maze testing, forced swimming, and combinations thereof.

22. The method of claim 20, wherein in step (2), the candidate substance is indicated as a potential therapeutic agent for a disease associated with spontaneous occurrence of mood swings when the behavioral analysis indicates a significant improvement in the symptoms of spontaneous occurrence of mood swings.