CN111004818A - A LGI 1 gene mutation and its application in the preparation of animal models of temporal lobe epilepsy comorbid depression - Google Patents
A LGI 1 gene mutation and its application in the preparation of animal models of temporal lobe epilepsy comorbid depression Download PDFInfo
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Abstract
The invention relates to the field of genetic engineering, in particular to a novel LGI1 gene mutation which is newly found in people and causes Temporal Lobe Epilepsy (TLE) and application thereof. The invention discloses an application of 152 th gene site A152G mutation of a No.1 exon of an LGI1 gene in preparation of a temporal lobe epilepsy co-morbid depression animal model and an application in screening of medicines for resisting temporal lobe epilepsy co-morbid depression. The invention discovers that the homozygous model mouse with gene mutation shows lethal epilepsy, the heterozygous mouse electroencephalogram can record epilepsy waveform, the ethology shows comorbid depression, and the homozygous model mouse can be used as a new animal model of epilepsy comorbid depression and used for mechanism research of epilepsy morbidity and epilepsy comorbid depression and research and development of anti-epilepsy comorbid depression medicines.
Description
Technical Field
The invention relates to a new LGI1 gene mutation which is newly discovered in people and causes Temporal Lobe Epilepsy (TLE), a homozygous model mouse with the gene mutation shows lethal epilepsy, an electroencephalogram of a heterozygous mouse can record epilepsy waveform, behavioristics shows comorbid depression, and the LGI1 gene mutation can be used as a new animal model of epilepsy comorbid depression and used for mechanism research of epilepsy pathogenesis and epilepsy comorbid depression and research and development of anti-epilepsy comorbid depression drugs.
Background
Temporal Lobe Epilepsy (TLE) is the most common focal epilepsy syndrome, and is one of partial seizure epilepsy, in which abnormal epileptic discharge often accompanied by pathological abnormalities such as hippocampus sclerosis, neuron loss, glial cell hyperplasia, moss fiber sprouting and the like originates from temporal lobe of brain. Temporal epilepsy is classified into Lateral temporal lobe epilepsy (L17 LE) and Medial Temporal Lobe Epilepsy (MTLE) [ 1 ] according to the location of the epileptogenic focus. Lateral temporal lobe epilepsy refers to epilepsy focus located in lateral neocortex of temporal lobe, including superotemporal gyrus, mediaotemporal gyrus, inferotemporal gyrus and fusiform gyrus; medial temporal lobe epilepsy refers to the epilepsy focus located in the medial temporal lobe structure, including hippocampus, amygdala and hippocampus.
Clinical studies found that about one third of epileptic patients have emotional disorders, called epilepsy co-morbidity, and in the last 70 th century, Trimble and Reynolds proposed for the first time the relationship of behavioral manifestations such as antiepileptic drugs and emotional cognitive disorders [ 2 ]. The results of a recent meta analysis showed that the correlation between the two reached 23.1% [ 3 ]. It has been clinically reported that up to 55% of patients with temporal lobe epilepsy suffer from combined depression and that the suicide mortality rate of temporal lobe epilepsy patients accompanied by depression is 25 times higher than that of patients with general epilepsy [ 4 ]. In addition to the pain associated with epileptic seizures, abnormal mental states also put great stress on patients.
Epilepsy-related depression is of increasing interest, but there is no uniform standard for clinical diagnosis or treatment. The diversity of depressive symptoms has led to difficulties in diagnosing epilepsy as a co-morbidity with depression. Kanner et al found that refractory epilepsy is associated with depressive patients, and that only 29% of patients have depressive symptoms that meet Diagnostic criteria for Mental Disorders and statistics IV (DSM), and diagnosis of depression requires professional Mental scale assessment, and there is no current method of assessment specifically for assessing epilepsy-related depression. In addition, because of the interference of side effects of taking antiepileptic drugs, the depression of epileptics can not be clear, and before the main target of controlling epileptic seizure, the improvement of depression symptoms is neglected. Therefore, the research on the pathogenesis of the temporal lobe epilepsy comorbid depression and the diagnosis and treatment have particularly important clinical significance.
Some clinical studies have shown, through long-term observation, an increase in depression following seizures [ 5 ], while studies on the other hand indicate that depressed individuals are at risk of developing epileptic patients [ 6 ]. This two-way correlation supports the hypothesis that there are overlapping mechanisms for epilepsy and depression, and may be mediated by genetic susceptibility. The role of genetic factors in seizures requires further intensive investigation.
To date, only one gene has a significant effect on the genetic susceptibility of TLE-the leucine-rich glioma inactivating 1 gene (LGI 1). A mutation in LGI1 was identified as approximately 50% in the family of autosomal dominant partial epilepsy with auditory function (ADPEAF), with an outbreak of approximately 67% [ 7 ]. By 2019, there were a total of 42 [ 8 ] loci of LGI1 that were reported in the ADTLE disease family. ADTLE is an idiopathic focal epilepsy syndrome with auditory symptoms or receptive facies as the major icteric manifestations. These symptoms strongly suggest a location of the lateral temporal lobe, and therefore the syndrome is also referred to as lateral temporal lobe epilepsy. Three people in the second generation of the LGI1c.431+1G > a mutant family exhibited psychiatric symptoms including depression and suicide [ 9 ] as shown in an ADPEAF family report with two mutation sites of LGI 1.
The temporal lobe epilepsy model is a very good precondition for researching the neurobiological mechanism related to depression of the epilepsy copropathies, until the research on genetic animal model is widely applied, an effective antiepileptic drug needs to be developed, and an important epilepsy development tool is provided for researching and developing a new clinical depression syngeneic epilepsy model, namely, a clinical antiepileptic drug with known clinical effect is used to prove the effective value of the model when an electric stimulation or chemical epilepsy inducing agent is used to copy the animal model, and then the model is used to evaluate the new antiepileptic drug, so that the selection of a proper animal model is very critical.
Reference to the literature
【1】Hesdorffer DC，Hauser WA，Annegers JF，Cascino G.Major depression isa 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-reportedepilepsyor seizure disorder and its associations with self-reporteddepressionand anxiety：results from the2004HealthStyles Survey.Epilepsia2006；47：1915-1921.
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Disclosure of Invention
The present invention investigates a new gene mutation known to have a strong impact on epilepsy risk and its risk for depressive symptoms. The invention aims to establish a novel epilepsy-combined depression mouse model based on pedigree data of epilepsy caused by 51 st amino acid mutation (D51G, aspartic acid mutation to glycine) in the 152 th gene site mutation (A152G) of the first exon (total 8 exons) region of LGI1 which is clinically found. Mice homozygous for this gene mutation exhibit refractory lethal epileptic characteristics with early onset. Although the mutation heterozygous mouse has no visible seizure epilepsy, the EEG test proves that the existence of the epilepsy wave is realized, the epilepsy onset age stage is later than that of the homozygous mouse, the epilepsy onset death is shown at different age stages, the depression phenotype is shown, the common genetic susceptibility hypothesis of the epilepsy and the depression is preliminarily shown, a suitable tool is provided for researching the neurobiological pathogenesis of the epilepsy and the comorbidity, and a new idea is provided for the research and development of the clinical epilepsy and depression combined medicine.
The invention discloses application of 152 th locus A152G mutation of A1 st exon in an LGI1 gene in preparation of an animal model of temporal lobe epilepsy co-morbid depression.
The invention discloses an application of a152 th gene site A152G mutation of an LGI1 gene exon in screening of a medicament for resisting temporal lobe epilepsy co-morbid depression.
The invention also discloses an animal model of temporal lobe epilepsy co-morbid depression, which is an ag heterozygote at the 152 th site of the No.1 exon of an LGI1 gene.
Further, the invention also provides a preparation method of the temporal lobe epilepsy co-morbid depression animal model, which comprises the following steps: designing an sgRNA recognition sequence, constructing an sgRNA, and constructing a corresponding donor vector according to the sgRNA; microinjecting a carrier to fertilized eggs and transplanting the fertilized eggs to superovulation receptor mice to obtain fountain mice, screening out positive fountain mice through PCR gene identification and sequencing, backcrossing the positive fountain mice and background mice to obtain F1 generations, mutually matching and breeding the F1 generations, then obtaining homozygous mutant mice through PCR primers and sequencing, and performing elevated plus maze and open field experiments on the homozygous mice in three months and six months and control wild mice to determine the depression phenotype.
The clinical data of the invention are derived from the basis of sorting and drawing a genealogical map of all personnel data of an inherited epilepsy (ADLTE) seizure family (the data is not published), extracting DNA from member blood samples, amplifying LGI1 exon regions, sequencing the amplified fragments, comparing the sequencing sequence with the sequence of a wild-type LGI1 genome exon region (ID: 56839) in GenBank, and screening out missense mutation (D51G) of 51 th amino acid in the first exon region of an LGI1 gene through comparison. Sequencing verification results show that the family members carrying the mutation are present in 4 generations of 34 members of the family, and 8 clinical diagnoses are ADLTE.
The model of the invention is prepared by constructing a mouse with Lgi1-D51G site mutation by using a Cas9/RNA system gene targeting technology on the basis of finding that a human LGI1 gene (ID: 9211) and an LGI1 gene (ID: 56839) of the mouse have high homology by comparison, and obtaining a homozygous or heterozygous Lgi1 gene mutation mouse by breeding. The survival characteristics and epileptic seizure characteristics of lgi1 mutant homozygous mice were continuously observed for 15 days from the 20 th postnatal day, and seizure levels were graded by Racine scoring method. Lgi1 the homozygous mouse with gene mutation has epileptic seizure in 3-5 weeks, the peak period of the seizure is 4 weeks or so, each seizure reaches five grades, and the seizure is characterized by jumping, falling down and strengthening the whole body. The interval period from the first onset to the death is about 8 hours on average, the attack frequency is shortened from 3 hours to 10 minutes, and the attack frequency from the first onset to the death is 3-5 times. The survival curves show that the genetically mutant mice show a longer survival time compared to lgi1 knockout mice.
According to the invention, EEG monitoring data of the heterozygous mice with gene mutation show that the brain waves of 75% of the heterozygous mice are abnormal and appear as high-frequency spike waves. Some mice died at different age stages. Three-month and six-month mice were subjected to the elevated plus maze and open field experiments to confirm that the heterozygous mice for more than six months had a depressive phenotype.
In conclusion, the invention firstly prepares the mouse with the mutation at the corresponding site according to the discovery of the new single site mutation of the LGI1 gene of epileptic patients, the homozygous mouse has the death of epileptic seizure, and the heterozygous mouse shows the depression caused by epileptic co-morbidity. The new mutation site homozygous model mouse provides a good model for researching a molecular mechanism of epilepsy pathogenesis, and the heterozygous mutation model mouse can become a good choice for researching a epilepsy comorbid depression mechanism.
Drawings
FIG. 1 is a survival curve for Lgi1 homozygous, heterozygous and wild mice.
FIG. 2 shows death (28 days) of Lgi1 homozygous mice after morbidity.
FIG. 3 shows death (344 days) of Lgi1 homozygous heterozygous mice after illness.
Figure 4 is a seizure waveform recorded in Lgi1 heterozygous mice (2 months).
FIG. 5 is the elevated plus maze for 3 months in Lgi1 heterozygous mice.
FIG. 6 is the elevated plus maze 6 months for Lgi1 heterozygous mice.
FIG. 7 is a3 month open field experiment of Lgi1 heterozygous mice.
FIG. 8 is a 6 month open field experiment of Lgi1 heterozygous mice.
Detailed Description
First, mouse model making
1. Design, construction and purification of vectors
(1) The strategy selects mice Lgi1-207(ENSMUST00000198518.4) to make, uses CRISPR Design tool (http:// CRISPR. mit. edu /) of the Massachusetts institute of technology, designs the target DNA with the length of 20bp according to the height of Score, the DNA sequence is shown as SEQ ID NO.1 (the underline position is mutation site a-g, simultaneously avoids the cleavage of knock-in sequence on Donor vector by cas9 protein, constructs Donor vector mutation site and makes a synonymous mutation site on exon1, the double underline position mutation is synonymous mutation c-a, g-t made on exon 1)
The oligonucleotide chain sequence of (a) is used for preparing an sgRNA sequence (the sgRNA sequence is SEQ ID NO. 2: CTGAGCCAACAGTGGTAGTA), a Donor vector carrying a target site homology arm and a target knock-in sequence is designed according to the sgRNA sequence, and a primer is designed in the target region for subsequent positive clone screening and gene identification of a mouse.
(2) The designed sequence was synthesized as a product of PAGE. Annealing the synthesized 2 single-stranded oligonucleotide sgRNA sequences (naturally cooling to room temperature after 5min at 95 ℃) to form double-stranded DNA, linking the double-stranded DNA with pGK1.1linear vector under the action of T4 DNA ligase, constructing a sgRNA expression vector, transforming the recombinant plasmid into DH5a competent cells, screening and identifying positive clone plasmids through kanamycin resistance of pGK1.1linear vector and sequencing of target DNA, selecting correct colony clone, and extracting plasmids after amplification culture for preparing an in vitro transcription template. Preparing a Donor vector fragment carrying a target site homology arm and a target knock-in sequence, connecting the Donor vector fragment with a T-vector, constructing a junction, constructing the Donor vector, transforming a recombinant plasmid into a DH5a competent cell, screening and identifying a positive clone plasmid through the ampicillin resistance of the T-vector and the sequencing of an insert fragment, selecting a correct colony clone, extracting the plasmid after amplification culture and purifying, and using an obtained Donor fragment product for injection.
2. Sample preparation and microinjection
(1) In vitro transcription of the sample: cas9 expression plasmid (Addgene No.44758), linearized by Age I digestion, purified by phenol chloroform extraction, dissolved in nuclease-free water as a template for in vitro transcription; cas 9mRNA was synthesized in vitro using T7 RNA polymerase according to the T7 Ultra Kit (Ambion, AM1345) Kit. The expression vector of the sgRNA is linearized by DraI enzyme digestion, extracted and purified by phenol chloroform, dissolved in nuclease-free water as a template for in vitro transcription, and synthesized into the sgRNA by T7 RNA polymerase outside a MEGAshortscript Kit (Ambion, AM1354) reagent Kit;
(2) microinjection of Cas9/sgRNA and donor: mixing the transcribed Cas 9mRNA, sgRNA and purified donor fragment, adjusting the concentration to 20 ng/ul Cas 9mRNA, 10 ng/ul sgRNA and 50 ng/ul donor fragment, microinjecting the mixture into the proto-nucleus and cytoplasm of fertilized egg of C57BL/6 mouse by TE2000U microinjection apparatus, transplanting the fertilized egg into the uterus of pseudopregnant C57 BL/6-mother mouse, and waiting for F0 mouse to be born; third, F0 mouse birth and identification and genetic detection in 5-7 days after F0 mouse birth, using a toe cutting method to mark the mouse, extracting DNA from cut rat tail tissue by a phenol chloroform method, identifying a primer (Forward: GACCTGTTCTTAGAGCAAGACAATC (SEQ ID NO. 3); Reverse: TGTTAGTGCTGTCAAATGGTCAGG (SEQ ID NO.4)) designed in a target region, and selecting a PCR positive sample for sequencing. The F0 generation mouse with correct PCR and sequencing is mated with a wild type C57BL/6 mouse to generate an F1 generation mouse, the F1 generation mouse is identified according to the identification method of the F0 generation mouse, and the obtained positive F1 generation heterozygote mouse can be stably inherited.
Second, detection of epilepsy in mice
All mice were scored for time of birth, identified by sequencing, genotyped and caged and then observed continuously and scored for time of death and seizure characteristics. The epileptic seizure strength adopts Racine grading standards (grade 0: no response; grade 1: facial muscle twitching, rhythmic chewing; grade 3: rhythmic nodding; grade 4: bilateral forelimb clonus with standing; grade 5: falling, generalized tonic clonus seizure). After anesthesia, the cells were fixed in a stereotaxic apparatus (512600, Stoelting, USA). Electrodes were implanted in the ventral hippocampal CA3 region (AP. -2.9 mm; L. -3.0 mm; V. -3.0mm) for electroencephalographic recording. In addition, a stainless steel screw is screwed into the skull above the sensory-motor cortex and is connected with an electrode for recording the electrocortical waves, and two screws are screwed into the skull above the cerebellum as the reference and grounding during the electrocerebral recording. The outer end of the electrode is welded on the micro socket and fixed on the skull by dental cement. All positional coordinates were measured with bregma as the origin, with reference to brain maps published by Franklin and Paxinos. After the behavioural experiment is finished, all mice need to confirm the electrode implantation position, and only the mice with correct electrode positions can be used for result analysis. The brain electrical activity of free-living mice was recorded using a PowerLab system (ADInstructions) at a sampling rate of 1kHz, 8 hours per day (9: 00 am. 5: 00 pm) and recorded for three consecutive days as a baseline. After the electroencephalogram recording is finished, the recorded EEG signals are analyzed off line by using LabChart 7 software in a PowerLab system. According to previous reports, epileptiform brain electricity is defined as: the time duration N is more than 20s, the peak frequency is more than or equal to 2Hz, and the amplitude is 3 times of that of the regular peak discharge clusters of the EEG base line. And 2 electroencephalogram attacks are separated by at least 10 s. In this experiment, spontaneous epileptic waves in 8-hour brain waves recorded every day were counted.
Third, Depression-related behavioural test
1. Elevated cross maze: the elevated plus maze consists of a plus maze part and a strut part. The cross-shaped part consists of two open arms, two closed arms and a central platform for connecting the open arms and the closed arms, wherein the length and the width of the open arms and the width of the closed arms are respectively 30cm multiplied by 5cm, the size of the central platform is 5cm multiplied by 5cm, the closed arms except the edges connected with the central platform are all provided with lightproof boards with the height of 15cm, so that dark areas are formed. The height of the support supporting the cross-shaped part is 38.5 cm. During the experiment, the overhead cross is placed on the flat ground, the camera for monitoring the movement of the mouse is vertically placed above the overhead cross, the head of the mouse is opposite to one of the open arms, and the camera is used for tracking and recording the movement of the mouse on the overhead cross. Each mouse was observed for 5min and the number of arm entries was used to calculate: number of open arms/(number of open arms + number of closed arms). The results were calculated for statistical analysis.
2. Open field experiment: the experimental environment is required to be closed and quiet, and the sound insulation of a laboratory is better, so that the influence of the external environment and the sound can be avoided. The size of the open box for the open field experiment in the laboratory is (50cm multiplied by 20cm) length multiplied by width multiplied by height, and the middle of the open box is a central area of 20cm multiplied by 20 cm. The central region is 10cm from the box edges on each side. The camera system is vertically arranged above the open box. During the experiment, the mouse is lightly placed at the center of the open field box, then the software is opened, the movement track of the mouse is collected for 20min, and the times of entering the open field center and the staying time of the mouse are recorded. After the experiment of each mouse, the residual odor of each mouse was removed by wiping with 75% ethanol to prevent the effect on the next mouse.
Fourth, statistical method
Statistical analysis is carried out by adopting SPSS19.0, the level of statistical significance is set to be less than or equal to 0.05, mean plus or minus standard deviation (mean plus or minus SD) is adopted, and the method of Leven's test is used for detecting normality and homogeneity of variance. If the normality and the homogeneity of variance are met, carrying out statistical analysis by using a T test (T-test); if the normality and variance are not met, the Kruskal-Wallis test is used. If the Kruskal-Wallis Test is statistically significant (P < 0.05), a comparative analysis is performed using Dunnett's Test (nonparametric method). Statistical differences and biological significance were considered for the evaluation.
Results of the experiment
Lgi1-D51G mutant mice survival curves and seizure characteristics
The disease characteristics of the obtained mutation homozygous mice are observed after gene identification, and 10 homozygous mice are recorded in total. Lgi1 the homozygous mice with gene mutation have epileptic seizure in 3-5 weeks, the peak period of the onset is 4 weeks or so (see figure 1), each onset reaches five grades, which is characterized in that one side falls down and then is maintained for about 5 seconds, then falls down and whole body spasticity, and the onset intensity reaches 5 grades. The interval period from the first onset to the death is about 8 hours on average, the attack frequency is shortened from 3 hours to 10 minutes, the attack frequency from the first onset to the death is 3-5 times, and the spasm state of limbs is maintained after the death along with the sudden death after the last onset (see figure 2). A total of 7 heterozygous mice were observed to die during the recording period, with the death times varying from 50 to 344 days, one of which had seizures observed upon cage change (see fig. 3), and the remaining dead mice had no observed seizures before death, but had spasticity in their limbs, which was identified as seizure death.
Lgi1-D51G mutant heterozygous mouse conscious epileptiform discharges
The electroencephalogram of the wild mouse is mainly expressed as a waves, the frequency is mainly expressed as 7-10HZ, the wave amplitude is mainly based on 20uv waves, a small amount of theta waves exist at the same time, and the a waves and the theta waves are basic waves and form the background activity of the electroencephalogram. 2 month old heterozygous mice tested continuously for 7 days showed explosive spikes and spikes on background activity (FIG. 4).
Lgi1-D51G mutant hybrid mouse elevated plus maze
Statistical analysis is respectively carried out on the frequency of the wild type and the gene mutation heterozygous mouse entering the closed arm for 3 months and 6 months, the result shows that no statistical difference exists between the elevated maze and the wild type of the heterozygous mouse for 3 months, the frequency of the mutation mouse entering the closed arm is obviously higher than that of the wild type mouse, the total movement distance is obviously reduced, the statistical significance is realized, and the anxiety behavior is shown (figure 5 and figure 6).
Lgi1-D51G mutant heterozygous mouse open field experiment
And (5) counting the moving speed, the total moving distance and the residence time of the central area of the mouse. Statistical results show that the residence time of heterozygous mice in the central area is significantly higher than that of normal mice. Lgi1 gene mutant mice were shown to exhibit depression-like behavior beginning at six months (FIG. 7, FIG. 8).
SEQUENCE LISTING
<110> Nanjing City health care hospital for women and children
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Cloud and stone
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