CN111004818B - LGI1 gene mutation and application thereof in preparation of temporal lobe epilepsy co-morbid depression animal model - Google Patents

Abstract

The invention relates to the field of genetic engineering, in particular to a new LGI1 gene mutation and its application newly discovered in the crowd that causes temporal lobe epilepsy (TLE). The invention discloses the application of the A152G mutation at the 152nd position of the first exon of the LGI 1 gene in the preparation of an animal model of temporal lobe epilepsy comorbid depression and the application in screening anti-temporal lobe epilepsy comorbid depression drugs. The present invention finds that the homozygous model mice with this gene mutation exhibit fatal epilepsy, the EEG of heterozygous mice can record epilepsy waveforms, and the behavior shows comorbid depression, which can be used as a new animal model of epilepsy comorbid depression. Research on the mechanism of epilepsy and epilepsy comorbid depression, and the research and development of antiepileptic drugs combined with depression.

Description

A LGI 1 gene mutation and its role in the preparation of an animal model of temporal lobe epilepsy comorbid depression application in

technical field

The present invention relates to a new LGI1 gene mutation newly discovered in the population that leads to temporal lobe epilepsy (TLE). Homozygous model mice with this gene mutation exhibit lethal epilepsy, and the EEG of heterozygous mice can record epileptic waveforms , Behavioral studies show comorbid depression, which can be used as a new animal model for epilepsy comorbid depression, for research on the mechanism of epilepsy and epilepsy comorbid depression, and for the development of antiepileptic drugs combined with depression.

Background technique

Temporal lobe epilepsy (TLE) is the most common focal epilepsy syndrome, often accompanied by pathological abnormalities such as hippocampal sclerosis, neuron loss, glial cell proliferation, and mossy fiber sprouting Abnormal epileptic discharges originate in the temporal lobe of the brain and are partial A type of epilepsy. Temporal lobe epilepsy is divided into lateral temporal lobe epilepsy (L17LE) and medial temporal lobe epilepsy (MTLE) according to the location of the epileptogenic focus [1]. Lateral temporal lobe epilepsy refers to the epileptogenic foci located in the neocortex of the lateral temporal lobe, including the superior temporal gyrus, middle temporal gyrus, inferior temporal gyrus, and fusiform gyrus; medial temporal lobe epilepsy refers to the epileptogenic foci located in the medial temporal lobe structures, including the hippocampus and amygdala nucleus and parahippocampal gyrus.

Clinical studies have found that about one-third of epilepsy patients have emotional disorders, which are called epilepsy comorbidities. In the 1970s, Trimble and Reynolds first proposed the relationship between antiepileptic drugs and behavioral manifestations such as emotional cognitive impairment [2]. The results of a recent meta-analysis showed that the correlation between the two reached 23.1% [3]. According to clinical reports, up to 55% of patients with temporal lobe epilepsy suffer from comorbid depression, and the suicide mortality rate of temporal lobe epilepsy patients with depression is 25 times higher than that of ordinary epilepsy patients [4]. In addition to the pain caused by epileptic seizures, the abnormal mental state of patients with epilepsy comorbidities also puts patients under tremendous pressure.

Epilepsy-related depression has received increasing attention, but there is still no uniform standard for clinical diagnosis or treatment. The diversity of depressive symptoms makes the diagnosis of epilepsy comorbid depression difficult. Kanner et al. found that in treatment-resistant epilepsy patients with depression, only 29% of the patients had depressive symptoms that met the diagnostic criteria of Diagnostic and Statistical Manual of Mental Disorders (DSM), and the diagnosis of depression requires professional psychological scales However, there is currently no specific assessment method for the assessment of epilepsy-related depression. Coupled with the interference of side effects of antiepileptic drugs, the depression of epilepsy patients is often unclear. Before the main goal of controlling epileptic seizures, improving depressive symptoms has become a neglected demand. Therefore, it is of great clinical significance to study the pathogenesis, diagnosis and treatment of depression with temporal lobe epilepsy.

Some clinical studies have confirmed the increase in depression after epileptic seizures through long-term observation [5], while studies on the other hand have shown that depressed individuals are at risk of developing epilepsy [6]. This bidirectional correlation supports the hypothesis that epilepsy and depression have overlapping mechanisms and may be mediated by genetic predisposition. Therefore, the role of genetic factors in epileptic seizures needs further in-depth study.

So far, only one gene has a significant impact on genetic susceptibility to TLE - the leucine-rich glioma inactivation 1 gene (LGI1). Mutations in LGI1 have been identified in approximately 50% of families with auditory autosomal dominant partial epilepsy (ADPEAF), with a penetrance of approximately 67% [7]. As of 2019, a total of 42 LGI1 mutation sites have been reported in ADTLE affected families [8]. ADTLE is an idiopathic focal epilepsy syndrome with auditory symptoms or a receptive phase as the predominant icteric manifestation. These symptoms strongly suggest a location in the lateral temporal lobe, so the syndrome is also known as lateral temporal lobe epilepsy. A report of an ADPEAF family with two LGI1 mutation sites showed that in the second generation of the LGI1c.431+1G>A mutation family, three people had mental symptoms including depression and suicide [9].

Temporal lobe epilepsy is an important class of diseases of the nervous system, and it is necessary to develop effective antiepileptic drugs. When designing and developing new antiepileptic drugs, animal epilepsy models must be used. Experimental animal models of epilepsy are similar to human epilepsy, and its mechanism is also close to the pathophysiological state of human seizures. When using electrical stimulation or chemical epilepsy agents to replicate animal models, generally known clinically effective antiepileptic drugs at that time are used to prove the effective value of the model, and then these models are used to evaluate new antiepileptic drugs, so appropriate animal models are selected very critical. An ideal temporal lobe epilepsy model should meet the following conditions: ① Symptomatically, the hippocampus, amygdala and other limbic structures occupy a central position; ② Pathologically, specific hippocampal damage is related to hippocampal sclerosis in temporal lobe epilepsy; ③ There is spontaneous Convulsions; ④ Like human temporal lobe epilepsy, it is resistant to most antiepileptic drugs. The ideal temporal lobe epilepsy model provides an excellent prerequisite for studying the neurobiological mechanisms related to epilepsy comorbidity and depression. To date, no specific genetic animal model has been widely used to study comorbidities of temporal lobe epilepsy. Therefore, it is of great clinical significance to study the role of lgi1 gene in the occurrence and development of epilepsy comorbid depression, and it also provides a new model tool for the drug development of epilepsy comorbid depression.

references

【1】Hesdorfffer DC, Hauser WA, Annegers JF, Cascino G. Major depression is a risk factor for seizures inolder adults. Ann. Neurol. 2000; 47: 246-249

【2】Trimble MR, Reynolds EH. Anticonvulsant drugs and mental symptoms: areview. PsycholMed1976; 6: 169-78.

【3】Kobau R, Gilliam F, Thurman DJ. Prevalence of self-reported epilepsy or seizure disorder and its associations with self-reported depression and anxiety: results from the 2004 Health Styles Survey. Epilepsia 2006; 47: 1915-1921.

【4】Mazza M. OrsucciF, DeRS, BriaP, MazzaS. Epilepsy and depression: riskfactors for suicide? Clin Ter 2004;155;425-7

【5】Jacoby A, Baker GA, Steen N, Potts P, Chadwick DW. The clinical course of epilepsy and its psychosocial correlates: findings from a U.K. Community study. Epilepsia. 1996; 37: 148-161

【6】Hesdorffer DC, Hauser WA, Annegers JF, Cascino G. Major depression is a risk factor for seizures inolder adults. Ann. Neurol. 2000; 47: 246-249

【7】Rosanoff MJ, Ottman R. Penetrance of LGI 1 mutations in autosomaldominant partial epilepsy with auditory features. Neurology. 2008; 71: 567-571

【8】Atsushi Yamagata, Yuri Miyazaki et al.Structural basis of epilepsy-related ligand-receptor complex LGI 1-ADAM22.Nature communications.2019

【9】Chabrol E, Popescu C, Gourfinkel-An I, Trouillard O, Depienne C, Senechal K, Baulac M, LeGuernE, Baulac S.Two novel epilepsy-linked mutations leading to a loss of function of LGI1.Arch.Neurol.2007 ;64:217-222.

Contents of the invention

The present study investigated a novel genetic mutation known to have a strong effect on epilepsy risk and its risk for depressive symptoms. The purpose of the present invention is to cause the 51st amino acid mutation (D51G, aspartic acid mutation to Glycine) induced epilepsy family data as a basis to establish a new epilepsy-combined depression mouse model. Mice homozygous for the mutation in this gene exhibit features of early-onset, refractory lethal epilepsy. Although the mutant heterozygous mice had no visible epilepsy, the EEG test confirmed the existence of epileptic waves. The age of onset of epilepsy was later than that of homozygous mice, and they showed onset and death at different ages, and showed a depression phenotype , preliminarily showed the common genetic susceptibility hypothesis of epilepsy and depression, provided a suitable tool for exploring the neurobiological pathogenesis of epilepsy and comorbidities, and provided new ideas for the development of drugs for clinical epilepsy combined with depression.

The invention discloses the application of the A152G mutation at the 152nd position of the first exon of the LGI 1 gene in preparing an animal model of temporal lobe epilepsy comorbid depression.

The invention discloses the application of the A152G mutation at the 152nd position of the exon of the LGI 1 gene in screening anti-temporal lobe epilepsy comorbid depression drugs.

The invention also discloses an animal model of temporal lobe epilepsy comorbid depression, which is ag heterozygous at position 152 of exon 1 of LGI 1 gene.

Further, the present invention also provides a method for preparing an animal model of temporal lobe epilepsy comorbid depression, comprising the following steps: designing the sgRNA recognition sequence, constructing the sgRNA, and constructing the corresponding donor vector according to the sgRNA; microinjecting the vector into the fertilized egg And transplanted to superovulated recipient mice to obtain Founder mice. Positive Founder mice were screened by PCR gene identification and sequencing, and positive Founder mice were backcrossed with background mice to obtain F1 generation. The homozygous mutant mice were obtained by sequencing, and the elevated plus maze and open field experiments were performed on the homozygous mice and control wild mice at three months and six months to determine their depression phenotype.

The clinical data of the present invention is derived from the data of all members of a genetic epilepsy (ADLTE) family and drawing a pedigree (the data has not yet been published). The blood samples of the members are extracted to extract DNA and amplify the exon region of LGI1. The amplified fragment was sequenced, and the sequencing sequence was compared with the sequence of the exon region of the wild-type LGI1 genome (ID: 56839) in GenBank, and an amino acid located at position 51 of the first exon region of the LGI1 gene was screened out by comparison. missense mutation (D51G). The results of sequencing verification showed that there were 34 members of the family carrying the mutation in 4 generations, and 8 of them were clinically diagnosed as ADLTE.

The model of the present invention is based on the high homology between the human LGI1 gene (ID: 9211) and the mouse lgi1 gene (ID: 56839), and the construction of the Lgi1- The mice with D51G site mutation were bred to obtain homozygous or heterozygous Lgi1 gene mutant mice. The survival characteristics and seizure characteristics of lgi1 mutant homozygous mice were observed continuously for 15 days from the 20th day after birth by continuous video recording, and the seizure levels were graded by Racine scoring method. Homozygous mice with the Lgi1 gene mutation developed epileptic seizures within 3-5 weeks, and the peak of the onset was around 4 weeks. The average interval from the first onset to death is about 8 hours, the frequency of attacks is shortened from 3 hours to 10 minutes, and the number of onsets from the first onset to before death is 3-5 times. Survival curves showed that compared with lgi1 knockout mice, the mutant mice showed a longer survival period.

According to the EEG monitoring data of the heterozygous mice with gene mutation, 75% of the heterozygous mice have abnormal brain waves, which are manifested as high-frequency spikes. Some mice died at different ages. Three- and six-month-old mice were subjected to elevated plus maze and open-field tests, which confirmed the presence of a depressive phenotype in heterozygous mice older than six months.

To sum up, the present invention is the first to produce mice with the corresponding point mutation based on the discovery of a new single point mutation of the LGI1 gene in the population of epileptic patients. Homozygous mice have epileptic seizures and lead to death, and heterozygous mice show epilepsy comorbidity and depression. The homozygous model mouse for the new mutation site provides a good model for studying the molecular mechanism of epilepsy, and the heterozygous mutant model mouse can be a good choice for studying the mechanism of epilepsy comorbid depression.

Description of drawings

Figure 1 is the survival curves of Lgi1 homozygous, heterozygous and wild mice.

Figure 2 shows the death of Lgi1 homozygous mice after onset (28 days).

Figure 3 shows the death of Lgi1 homozygous heterozygous mice after onset (344 days).

Figure 4 is the epileptic seizure waveform recorded in Lgi1 heterozygous mice (2 months).

Figure 5 is the elevated plus maze of Lgi1 heterozygous mice at 3 months.

Figure 6 is the elevated plus maze of 6-month-old Lgi1 heterozygous mice.

Figure 7 is the open field experiment of Lgi1 heterozygous mice at 3 months.

Figure 8 is the open field experiment of Lgi1 heterozygous mice at 6 months.

Detailed ways

1. Mouse model making

1. Design, construction and purification of vectors

的寡聚核苷酸链序列用于制备sgRNA序列(sgRNA序列为SEQ ID NO.2：CTGAGCCAACAGTGGTAGTA)，根据sgRNA序列设计一个携带靶位点同源臂以及目的敲入序列的Donor vector，并在该靶区域设计引物用于后续阳性克隆筛选以及小鼠的基因鉴定。(1) In this strategy, mouse Lgi1-207 (ENSMUST00000198518.4) was selected for production, using the CRISPR Design tool of the Massachusetts Institute of Technology (http://crispr.mit.edu/), and a 20bp target was designed according to the level of Score Target DNA, DNA sequence such as SEQ ID NO.1 (the underlined position is the mutation site ag. At the same time, in order to prevent the cas9 protein from cutting the knock-in sequence on the Donor vector, construct the Donor vector mutation site and make a synonym on exon1 The mutation site, the double underlined position is mutated to a synonymous mutation made on exon1 (ca, gt)

The oligonucleotide chain sequence of the sgRNA is used to prepare the sgRNA sequence (the sgRNA sequence is SEQ ID NO.2: CTGAGCCAACAGTGGTAGTA), and a Donor vector carrying the homology arm of the target site and the target knock-in sequence is designed according to the sgRNA sequence, and in the Primers were designed for the target region for subsequent screening of positive clones and gene identification of mice.

(2) Synthesize the designed sequence into a PAGE product. The synthesized two single-stranded oligonucleotide sgRNA sequences were annealed (95°C for 5 minutes and then naturally cooled to room temperature) to form double-stranded DNA, which was linked to pGK1.1linearvector under the action of T4 DNA ligase to construct the sgRNA expression vector and recombine Transform the plasmid into DH5a competent cells. Through the kanamycin resistance of pGK1.1linearvector and the sequencing of the target DNA, the positive clone plasmids are screened and identified, the correct colony clones are selected, and the plasmids are extracted after expansion for in vitro transcription. template. Make the Donor vector fragment carrying the homology arm of the target site and the target knock-in sequence and connect it to the T-vector, construct the graft, construct the Donor vector Donorvector, transform the recombinant plasmid into DH5a competent cells, and pass the ampicillin resistance of the T-vector As well as the sequencing of insert fragments, screening and identification of positive clone plasmids, selection of correct colony clones, extraction of plasmids and purification after expansion and cultivation, and the obtained donor fragment products are used for injection.

2. Sample Preparation and Microinjection

(1) Sample in vitro transcription: Cas9 expression plasmid (Addgene No.44758), linearized by Age I digestion, extracted and purified with phenol chloroform, dissolved in nuclease-free water as a template for in vitro transcription; according to T7 Ultra Kit (Ambion, AM1345) kit, using T7 RNA polymerase to synthesize Cas 9 mRNA in vitro. The sgRNA expression vector was linearized by DraI digestion, extracted and purified with phenol chloroform, dissolved in nuclease-free water as a template for in vitro transcription, and passed T7 RNA polymerase in vitro according to the MEGAshortscript Kit (Ambion, AM1354) kit Synthetic sgRNA;

(2) Microinjection of Cas9/sgRNA and donor: mix transcribed Cas9 mRNA, sgRNA and purified donor fragments and adjust the concentration to 20ng/μl of Cas9 mRNA, 10ng/μl of sgRNA and 50ng/μl of donor Fragment, use the TE2000U microinjector to microinject the mixture into the pro-nucleus and cytoplasm of C57BL/6 mouse fertilized eggs, and then transplant the fertilized eggs into the uterus of pseudopregnant C57BL/6-mice, waiting for the F0 generation Birth of mice; 3. Birth and identification of F0 generation mice and genetic testing 5-7 days after the birth of F0 generation mice, the mice were marked with the toe cutting method and DNA was extracted by cutting the mouse tail tissue through the phenol-chloroform method. Primers (Forward: GACCTGTTCTTAGAGCAAGACAATC (SEQ ID NO.3); Reverse: TGTTAGTGCTGTCAAATGGTCAGG (SEQ ID NO.4)) designed in the target region were identified, and PCR-positive samples were selected for sequencing. The F0 generation mice with correct PCR and sequencing were mated with wild-type C57BL/6 mice to generate F1 generation mice, and the F1 generation mice were identified according to the identification method of F0 generation mice, and the positive F1 generation heterozygotes obtained Mice can be genetically stable.

2. Detection of epilepsy in mice

The time of birth, sequencing identification and genotype of all mice were recorded, and the death time and seizure characteristics of the mice were continuously observed and recorded after they were separated into cages. Seizure intensity was graded according to the Racine scale (grade 0: no response; grade 1: facial muscle twitching, rhythmic chewing; grade 3: rhythmic nodding; grade 4: bilateral forelimb clonus with standing; grade 5: fall, general rigidity clonic seizures). After anesthesia, they were fixed in a stereotaxic apparatus (512600, Stoelting, USA). Electrodes were implanted in the CA3 region of the ventral hippocampus (AP.-2.9mm; L.-3.0mm; V.-3.0mm) for EEG recording. In addition, a stainless steel screw was screwed into the skull above the sensorimotor cortex and connected to electrodes for recording cortical EEG, and two screws were screwed into the skull above the cerebellum as reference and grounding for EEG recording. The outer ends of the electrodes are welded to micro-sockets and fixed to the skull with dental cement. All positioning coordinates were measured from the bregma as the origin, referring to the brain atlas published by Franklin and Paxinos. After the behavioral experiment, all mice need to confirm the electrode implantation position, and only the mice with the correct electrode position can be used for the result analysis. The EEG of freely moving mice was recorded with the PowerLab system (ADInstruments) at a sampling rate of 1 kHz for 8 hours a day (9:00 am to 5:00 pm), and recorded for three consecutive days as a baseline. After the EEG recording was completed, the recorded EEG signals were analyzed offline with the LabChart 7 software in the PowerLab system. According to previous reports, epileptiform EEG is defined as regular spike discharge clusters with duration N>20s, spike frequency ≥2Hz, and amplitude 3 times the EEG baseline. At least 10 seconds between two EEG attacks at the same time. In this experiment, the spontaneous epileptic waves in the 8-hour EEG recorded every day were counted.

3. Depression-related behavioral testing

1. Elevated plus maze: The elevated plus maze consists of two parts: the plus maze part and the pillar part. The cross part is composed of two open arms, two closed arms and a central platform connecting the open arms and closed arms. On the side connected to the central platform, the other three sides have opaque wooden boards with a height of 15cm to create a dark area. The pillars supporting the cross section are 38.5cm high. During the experiment, the elevated cross was placed on the flat ground, and the camera for monitoring the movement of the mice was placed vertically above the elevated cross so that the head of the mouse faced one of the open arms, and the camera was used to track and record the activities of the mice on the elevated cross. Each mouse was observed for 5 minutes, and the number of times of entering the arm was used to calculate: number of open arms/(number of open arms+number of closed arms). The calculated results were used for statistical analysis.

2. Open-field experiment: The experimental environment is required to be closed and quiet, and it is best to make the laboratory soundproof, so as to avoid the influence of the external environment and sound. The size of the opening box used for the opening experiment in this laboratory is (50cm×50cm×20cm) length×width×height, and the middle of the opening box is the central area of 20cm×20cm. The central area is 10 cm from the edge of the box on each side. The camera system is placed vertically above the opening box. During the experiment, the mice were gently placed in the center of the opening box, and then the software was turned on to collect the mouse movement trajectory for 20 minutes, and the number of times the mice entered the center of the opening box and the time of staying were recorded. After the end of the experiment, each mouse was wiped with 75% ethanol to remove its residual odor to prevent it from affecting the next mouse.

4. Statistical methods

SPSS 19.0 was used for statistical analysis, and the level of statistical significance was set at P≤0.05. The mean ± standard deviation (mean ± SD) was used, and the normality and homogeneity of variance were tested by Leven's test method. If normality and homogeneity of variances were met, T-test was used for statistical analysis; if normality and variances were not met, Kruskal-Wallis test was used. If the Kruskal-Wallis test was statistically significant (P<0.05), Dunnett's Test (non-parametric method) was used for comparative analysis. Statistical differences and biological significance were considered in the evaluation.

Experimental results

Survival curve and seizure characteristics of Lgi1-D51G mutant mice

After genetic identification of the acquired mutant homozygous mice, the disease characteristics were observed, and a total of 10 homozygous mice were recorded. All homozygous mice with Lgi1 gene mutations developed epileptic seizures within 3-5 weeks, and the peak of the onset was around 4 weeks (see Figure 1). Seconds or so, he jumped, then fell down and had tonic convulsions all over his body, and the intensity of the attack reached level 5. The average interval between the first onset and death is about 8 hours, and the frequency of attacks is shortened from 3 hours to 10 minutes. The number of onsets from the first onset to death is 3-5 times, accompanied by sudden death after the last onset, and limb spasm after death state (see Figure 2). A total of 7 heterozygous mice died during the recording period, and the death time ranged from 50 days to 344 days. Among them, one mouse was observed to have seizures when changing cages (see Figure 3), and the remaining dead mice were not observed to die Before the epileptic seizure, but the limbs were in a state of tonic spasm, it can be determined that the death was due to epileptic seizures.

Awake epileptiform discharges in mice heterozygous for the Lgi1-D51G mutation

The EEG of wild-type mice mainly shows a wave, the frequency is mainly 7-10HZ, the amplitude is mainly 20uv waveform, and there is a small amount of θ wave at the same time. The 2-month-old heterozygous mice, which were continuously detected for 7 days, showed explosive spikes and sharp waves on the basis of background activities (Figure 4).

Elevated plus maze in mice heterozygous for the Lgi1-D51G mutation

Statistical analysis was performed on the frequency of entry into the closed arm of wild-type and mutant heterozygous mice at 3 months and 6 months respectively. Mice entered the closed arms significantly more frequently than wild-type mice, had a statistically significant reduction in the total distance traveled, and exhibited anxiety-like behavior (Fig. 5, Fig. 6).

Open field experiment in Lgi1-D51G mutant heterozygous mice

The mouse movement speed, the total movement distance and the dwell time in the central area were counted. Statistical results showed that the residence time of heterozygous mice in the central area was significantly higher than that of normal mice. It was shown that the Lgi1 gene mutant mice began to exhibit depression-like behaviors at six months (Fig. 7, Fig. 8).

SEQUENCE LISTING

<110> Nanjing Maternal and Child Health Hospital

Zhengfeng, Xu

Flat Hu

cloud stone

<120> A LGI 1 gene mutation and its application in the preparation of an animal model of temporal lobe epilepsy comorbid depression

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<160> 4

<170> PatentIn version 3.3

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Claims (2)

1. Application of 152G mutation heterozygote at 152 th locus A152G of exon1 of LGI1 gene in preparation of an animal model of temporal lobe epilepsy co-morbid depression.
2. Application of 152G mutation heterozygote at 152 th locus A152G of 1 st exon of LGI1 gene in screening of medicine for treating depression caused by temporal lobe epilepsy co-disease.

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