Construction of an active immune model and electrophysiological study of mice with anti-NMDAR encephalitis

doi:10.21203/rs.3.rs-1509233/v1

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Construction of an active immune model and electrophysiological study of mice with anti-NMDAR encephalitis

https://doi.org/10.21203/rs.3.rs-1509233/v1

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Background: anti-N-methyl-D-aspartate receptor encephalitis is an autoimmune disease, which can show severe neuropsychiatric symptoms, of which seizures are one of the most common symptoms. At present, the mechanism of anti-NMDAR encephalitis is still unclear, and the appropriate animal model is the basis of scientific research.

Methods: C57BL/6 male mice were actively immunized with the amino terminal domain peptide of GluN1 subunit. C57BL/6 mice were divided into GluN1_359-378group, control group and blank group. Electroencephalogram system was used to record rats' EEG. Indirect ELISA was used to detect serum NMDAR-AB after 14 days of ATD peptide active immunization. HEK293 cells containing GluN1 and GluN2B subunit plasmids were incubated with positive serum to detect NMDAR-AB；Hippocampal neurons were incubated with the serum of the above groups, and NMDAR-AB was detected. Immunohistochemistry was used to verify the serum NMDAR-AB of mice. Western blot was used to verify NMDAR-AB, Glutamic vesicle transporters and γ -Aminobutyric acid vesicle transporters in hippocampus.

Results: 1. 2 weeks after active immunization with ATD peptide, the detection rate of NMDAR-AB in serum of GluN1_359-378group was 67%, which was significantly higher than that of control group；2. The transfected HEK293 cells, hippocampal neurons and mouse brain sections were incubated with NMDAR-AB positive serum. The results showed that GluN1_359-378 peptide could induce serum autoantibodies against GluN1 and GluN2b; 3. In GluN1_359-378 peptide immunized mice, anti glun1 protein was mainly expressed in hippocampal neurons; 4. In GluN1_359-378 group, the expression of glun1 on the membrane surface decreased significantly. The decrease of glun1 on the membrane surface was related to the decrease of NMDAR cluster density on the surface of hippocampal neurons; 5. The total GluN1 of GluN1_359-378 group had no significant change, but the vGluT1 of hippocampal tissue increased and vGAT did not change. 6. Epileptic discharge was recorded in 78% of the GluN1_359-378mice, but not in the control group, which was related to the increase of vGluT1 in hippocampus.

Conclusion: the anti-NMDAR encephalitis model was successfully established by active immunization with GluN1_359-378 peptide targeting GluN1 gene ATD, and the mouse EEG showed epileptiform discharge.

anti-NMDA receptor encephalitis

GluN1

active immunity

serum

seizures

The anti-NMDAR encephalitis model was successfully established by active immunization with GluN1_359-378 peptides targeting the GluN1 gene ATD.
The results showed that 78% of the mice in the GluN1_359-378 group experienced epileptic discharges.
The expression of vGluT1 was increased in the GluN1_359-378 group.

Anti-NMDAR encephalitis is an autoimmune disease of the central nervous system (CNS), first identified in women with encephalitis and ovarian teratoma[1–3]. Typical clinical manifestations include progressive neurological and mental symptoms, including abnormal movements, seizures, memory disorders, and behavioral [4]. Among them, epilepsy is one of the most typical symptoms. Indeed, 80.9% of the patients reported epilepsy as their first or most prominent symptom [4]. NMDAR-Ab is positive in the cerebrospinal fluid and serum of patients with anti-NMDAR encephalitis[5]. Some studies have hypothesized that after immune activation is triggered by tumor-associated antigens or other mechanisms, the synthesized antibodies may cross the blood-brain barrier through the leakage area[6], while activated B cells may also cross the BBB into the brain and differentiate into plasma cells, continuously inducing NMDA receptor antibody synthesis in the sheath[7]. In contrast, other studies have reported that antibodies against NMDA receptor subunit GluN1 specifically alter the structure or function of synaptic receptors and down-regulate NMDAR levels[8] ; this is also considered a key mechanism of anti-NMDAR encephalitis[9]. However, the pathogenesis of anti-NMDA receptor encephalitis, especially epileptic seizures, remains unclear[10]. In previous experimental studies of passive transport in mice[11] and intracerebroventricular injection of patient antibody IgG in rats[12], the passive immunity of experimental animals exhibited depression-like behavior, pleasure deficiency, and memory deficit behavior[13]. These experimental studies proved the pathogenicity of NMDA receptor antibodies but could not simulate the production of autoantibodies in vivo[14]. Therefore, an active immune model closer to disease progression was required to explore the pathogenesis of anti-NMDA receptor encephalitis, especially regarding the mechanism of seizures[15]. An active immune animal model can simulate the production of antibodies in autoimmune diseases and plays a critical role in the study of the pathogenesis of autoimmune encephalitis[16, 17]. Studies have demonstrated that autoreactive antibodies against NMDAR can recognize part of the N-terminal domain (ATD) of NMDAR[7], including the key amino acids asparagine and glycine at position 368/369, which can amplify the immune effect of the disease[18]. Herein, we aimed to immunize mice with an ATD polypeptide for 2 weeks and assess whether the anti-NMDA receptor encephalitis model was successfully constructed by analyzing serum NMDAR-Ab, antibody expression in the hippocampus, and epileptic discharge via EEG.

Animal

Twenty-seven clean C57BL/6 mice, male aged 8 weeks old and weighing 20-25 g, were randomly assigned to either the GluN1_359-378group, control group, or blank group, with 9 mice in each group. They were purchased from Sbeifu (Beijing) Biotechnology Co., Ltd. [SCXK (Beijing) 2019-0010], license number: SCXK Gui 2020-0003, housed in the animal room of the 104th Museum of Guangxi Medical University, 12:12h light/dark diurnal cycle, and food and water ad libitum. After the experiment, the mice were killed by deep anesthesia with 3% Pentobarbital Sodium (30 mg / kg). The study was conducted in accordance with the "Guiding Opinions on the treatment of Laboratory Animals" issued by the Ministry of Science and Technology of the People's Republic of China and Guidelines for Ethical Review of laboratory Animal Welfare issued by the People's Republic of China (National Standard GB/T35892-2018).

Peptide design

Wagnon I, Helie P, et al. showed that the amino acids N368 and G369 on GluN1 can amplify the immune effect of anti-NMDAR encephalitis. In this study, the immunogenic peptide GluN1_359-378 was selected (RKLVQVGIYNGTHVIPNDRK) from the Nanjing peptide industry[13] .

Research design and active immunity in mice

8-week-old male C57BL/6 mice were immunized with an emulsifier mixed with complete Freund's adjuvant (CFA, Sigma Aldrich Laboratories) and GluN1_359-378. CFA contained Mycobacterium tuberculosis H37Ra (3 mg/ml). The volume ratio of CFA and GluN1_359-378 was 1:1, and the final peptide concentration was 1 mg/ml. The mice were subcutaneously injected with an antigen emulsifier at a dose of 200 ul/ to induce an autoimmune response. The control group was given an emulsion mixture of CFA and an equal volume of phosphate-buffered saline (PBS). 50 ml of the emulsion was injected four times: twice in the shoulder and twice in the limbs. Mice in the blank group were injected with the same amount of normal saline at the same positions. GluN1_359-378 and negative control group animals were intraperitoneally injected with 200 ul saline containing 200 ng pertussis toxin (Sigma Aldrich) on the day of immunization, and 48 hours after immunization, the mice in the blank group were injected with the same amount of normal saline.

Research and design

Animals were randomly allocated to experimental group: each cage was given a letter by an individual not associated with the experiments. The experimenter was blinded to experimental groups until the end of the protocol. The order in which the animals in the different experimental groups were treated and assessed was also determined in a random way at the beginning of the protocol. Correspondence between letters and experimental groups was revealed to the experimenter after all analyses were performed. Animal behavior was observed blindly every day, and the EEG was performed by a Nuocheng EEG recorder. On the 14th day, serum and hippocampal tissues of the mice were taken for further molecular biology investigations.

EEG recording

After the mice were anesthetized with 2,2,2-tribromoethanol, they were fixed with a stereotaxic instrument, with electrodes installed on their forehead, temporal, and forelimbs, and the EEG was recorded by the Nuocheng EEG system. Each site was marked as follows: left frontal Fp1-Avf, right frontal Fp2-Avf, left temporal T3-Avf, and right temporal T4-Avf. Paper speed 4.5 cm/s, sensitivity 200 uV/cm.

ELISA

Follow the instructions of the kit (Jianglai Biology, JL20420).

Preparation and staining of HEK293 cells expressing GluN1

According to the instructions of the NanoTrans 20TM transfection reagent in vitro (B1001PowerBiomedical), the plasmids of GluN1 and GluN2B subunits (Grin1, Grin2, Shanghai Genechem Co., Ltd.) were transferred into HEK293 cells, and the cell slides were broken with 4% paraformaldehyde for 30 minutes, and 0.3% Triton X-100 infiltrate for 5 minutes. After three 1-minute washes with PBS, the cells were stained using the four-color multiple fluorescence immunohistochemical staining kit (abs50012, Absin) according to the manufacturer’s instructions. Afterward, the cells were divided into 5 groups: In the CMV-NC+Serum（GluN1_359-378）group, HEK293 cells without plasmids were incubated in a positive serum; in the CMV-Grin1+ serum（control）group, HEK293 cells transfected with GluN1 plasmids were incubated in a negative serum; in the CMV-Grin1+Serum (GluN1_359-378) group, HEK293 cells transfected with GluN1 plasmids were incubated in a positive serum; in the CMV-Grin2+Serum (control) group, HEK293 cells transfected with GluN2B plasmids were incubated in a negative serum; in the CMV-Grin2+Serum (GluN1_359-378) group, HEK293 cells transfected with GluN2B plasmids were incubated in a positive serum, the antibodies were mouse negative serum (diluted 1:10), mouse positive serum (diluted 1:10), antibodies GluN1 (1:1000, Boster) and GluN2b (1:1000, SANGON biotech).

Primary hippocampal neuron culture

The hippocampus of newborn SD rats was sliced into pieces within 24 hours by tissue shearing and digested with 4 ml trypsin for 12 minutes. After the termination of digestion, a 4 ml implantation medium was added. After 1000× g centrifugation for 5 minutes, the cells were counted and then inoculated on a 24-well plate treated with poly D-lysine. After 24 h of culture, the supernatant was discarded and replaced with a 500 μl fresh maintenance medium. Every 3 days, half of the volume of the medium was removed from the cells, half of the volume of the medium was replaced, and the culture lasted 7 days.

Immunocytochemistry

The mice were divided into the GluN1359-378 peptide, negative control, and blank groups. Two weeks after active immunization with the ATD peptide, the hippocampal neuron cells were incubated with mouse serum, PBS, and antibody GluN1 (1: 50, BOSTER), and neuron-specific enolase (NSE) and NMDAR-Ab was detected. The cover glass of hippocampal neuron cells was washed with PBS 3 times for 5 minutes each time. Afterward, the cells were fixed with 4% paraformaldehyde for 30 minutes and rewashed with PBS 3 times for 5 minutes each time. After drying at room temperature, they were permeabilized with 0.2% Triton X-100 for 5 minutes and washed with PBS again. For the brain tissue of mice, the brains of untreated blank mice were processed into 5 μm thick paraffin sections. After deparaffinization and rehydration, the slices were repaired by enzyme digestion. The cells and brain slices were blocked and treated with the antibody enzyme-linked Goat anti-mouse IgG polymer according to the instructions of the two-step mouse Kit (Beijing Zhongshan Jinqiao Biotechnology Co., Ltd.), then stained with DAB (Beijing Zhongshan Jinqiao). Afterward, the slices were dehydrated and stained with hematoxylin. Finally, the sections were visualized using a Leica light microscope and recorded by Image View.

Experiment of protein immunoblotting technique

Membrane protein and total protein were extracted from the hippocampus by a microplasma membrane protein separation kit (invent). After the protein concentration was determined, 40 microliters of the membrane protein sample were mixed with 10 microliters of sodium dodecyl sulfate (SDS) sample buffer and subsequently boiled at 95 °C for 10 min. The samples were separated by 12.5% SDS polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes. After 12 minutes of sealing with rapid sealing fluid (Beyotime), they were incubated overnight with the primary antibodies GluN1 (1:1000， BOSTER), membrane protein internal reference ATP1A1 (1:1000,proteintech), vGluT1 (1:1000, Sangon), vGAT (1:1000, Sangon) and GAPDH (1:8000, Sangon) at 4 °C. After washing 3 times with TBST, the membranes were incubated with goat anti-rabbit IgG (1:8000, Sangon Biotech) at room temperature for 1 hour. Finally, the immune complex was developed using a hypersensitive chemiluminescence kit (enhanced) (Thermo Science).

Statistical analysis

Statistical analyses were performed by the SPSS23.0 statistical software package, and all data were expressed by mean±Standard Deviation (Mean±SD). An independent sample T-test or nonparametric test was applied to assess the significance of differences between the two data groups, analysis of variance were used to compare differences between different groups. All figures were constructed using GraphPadPrism6.0 (GraphPad Software, USA) or ImageJ. P < 0.05 was considered statistically significant.

Qualitative detection of serum NMDAR-Ab in mice immunized with GluN1_359-378 peptide

To verify the pathogenicity of the antigenic peptide, NMDAR-AB was qualitatively detected by ELISA in the serum of each group of mice. The positive rate was 67% in the GluN1_359-378 group, 11% in the control group, and 0 in the blank group. ELISA results demonstrated that the positive rate in the GluN1_359-378 group was significantly higher than that of the control and blank groups, P<0.05(Figure 1).

Determination of NMDAR-Ab receptor GluN1 and GluN2B subunits in the serum of mice immunized with the GluN1_359-378 peptide

In order to confirm the presence of pathogenic antigen peptides in the serum, HEK293 cells transfected with GluN1 and GluN2B subunits were incubated with a positive serum, and green fluorescence was visualized under a confocal microscope (Figure 2a). The transfection efficiency of HEK293 cells was evaluated by WB. The results revealed that GluN1 was significantly expressed in the CMV-Grin1 group (P<0.05) (Figure 2b), whereas GluN2B was significantly expressed in the CMV-Grin2 group (P<0.05) (Figure 2c), indicating that transfection was successful. HEK293 cells transfected with GluN1 and GluN2B subunits were incubated with a positive serum and negative serum, respectively, and red fluorescence was observed under a confocal microscope (Figure 2a). The results indicated that only the GluN1_359-378 peptide could induce serum autoantibodies against GluN1 and GluN2B

Expression of Anti-GluN1 protein in Hippocampal neurons of mice immunized with the GluN1_359-378 Peptide

The hippocampal neurons of SD suckling mice were incubated with an NMDAR-Ab positive mouse serum immunized with the GluN1_359-378 peptide and NSE, respectively. Under a fluorescence microscope, red fluorescence, green fluorescence, and mixed yellow fluorescence were identified in the cell bodies and axons of hippocampal neurons in the GluN1_359-378 group, but no red fluorescence was found in hippocampal neurons incubated with serum in the control and blank groups (Figure 3a).

Immunohistochemical staining showed prominent brown deposits on the surface of hippocampal neurons in the GluN1 antibody group and NMDAR-AB positive group immunized with the GluN1_359-378 peptide (Figure 3b).

The brains of mice in the blank group were decapitated and cut into brain slices, which were incubated with a positive serum and the GluN1 antibody of the GluN1_359-378 group, respectively. The results showed prominent positive brown deposits on the surface of hippocampal and cortical neurons incubated with the positive serum of the GluN1_359-378 group. However, no positive staining was detected in the negative sera of the GluN1_359-378, control, blank groups, and the brain slices incubated with PBS of the negative control group (Figure 3c, d).

Furthermore, the results showed that NMDAR-Ab induced by the GluN1_359-378 peptide was expressed in hippocampal neurons and the hippocampal cortex.

GluN1_359-378 antibody reduces the expression of NMDA receptor GluN1 protein on the cell membrane.

The expression of GluN1 protein in the hippocampus of mice immunized with the GluN1_359-378 peptide was detected by WB. The results revealed that the expression of GluN1 on the membrane surface of the GluN1_359-378 group was significantly decreased (P<0.05) (Figure 4a), but the total tissue protein GluN1 remained unchanged (P>0.05) (Figure 4b).

Electroencephalogram of the GluN1_359-378 Peptide immunized mice

No apparent epileptic symptoms were observed in the mice immunized with the GluN1_359-378 peptide. The EEG of mice was recorded for 30 minutes a day by an EEG system. The results showed that 78% of the mice in the GluN1_359-378 group experienced epileptic discharges, while no epileptic discharges were recorded in the control group (Figure 5).

Increased nerve excitability in mice immunized with the GluN1_359-378 peptide

Furthermore, the cause of epileptic discharge in EEG of mice immunized with the GluN1_359-378peptide was explored, and VGlut1 (glutamate vesicle transporter 1) and γ-aminobutyric acid (GABA)-responsive vGAT (γ-aminobutyric acid vesicle transporter) were detected. WB was employed to analyze the expression of vGluT1 and vGAT in the hippocampal tissues of mice immunized with the GluN1_359-378 peptide for 2 weeks. The results illustrated that the expression of vGluT1 was increased in the GluN1_359-378 group (P<0.05), but there was no significant change in vGAT (P>0.05) (Figure 6).

This study detailed the active immunization against the GluN1_{359 − 378} peptide in mice, and our results demonstrated that this polypeptide could induce and maintain the expression of pathogenic GluN1 autoantibodies in mice.

First of all, by consulting the literature, we screened and identified the possible antigenic targets of anti-NMDA receptor antibodies, designed antigenic peptides for in vivo induction, and evaluated the expression of pathogenic anti-GluN1 autoantibodies in the serum of mice after immunization via several methods.

The results of the ELISA test were inconsistent; 33% of the GluN1_{359 − 378} group sera were negative, and 11% of the control group sera were positive. A possible explanation may be that when using ELISA to detect antibodies, the coated antigens should contain as many specific antigenic determinants as possible while discluding as few non-specific components as possible, which is often challenging to achieve owing to technical limitations. Therefore, in a sense, the detection of false-positive and false-negative pathogenic antibodies in some sera can not be avoided. Indeed, false positives and false negatives have been reported in the detection of serum antibodies by ELISA[19]. On the other hand, this may be related to the low titer of pathogenic antibodies in the serum. It has been previously reported that the results of ELISA may be negative when the antibody titer in serum is too low[20], which is consistent with the results of the positive cerebrospinal fluid test, epileptic EEG discharge, and negative serum tests in patients with anti-NMDAR encephalitis reported by Josep Dalmau et al.[1]. According to the transfection, immunohistochemistry, and WB results, false positives and false negatives caused by experimental errors were considered. Due to the small size of C57BL/6, not easy to fix, resulting in high mortality in the process of collecting cerebrospinal fluid, too little amount of cerebrospinal fluid, and other reasons, this study can not be verified by cerebrospinal fluid.

The HEK-293 cells transfected with the NMDAR subunit were incubated with serum, and the expression of anti-NMDAR autoantibody (NMDAR-Ab) in blood was analyzed. The results showed pathogenic anti-GluN1 autoantibodies in the positive serum of the GluN1_{359 − 378} group. The brain slices of mice in the blank group incubated with the serum were detected by immunohistochemical staining. The results confirmed that the antibodies produced by the mice immunized with the GluN1359-378 peptide were deposited on the surface of neurons in the hippocampus and cortex. These findings are consistent with the immune binding characteristics of antibodies derived from the cerebrospinal fluid of patients with anti-NMDAR encephalitis on rat brain sections[21]. Western Blot was used to detect the hippocampus of mice. The results showed that while the antibodies produced in response to immunization with the GluN1_{359 − 378} peptide did not affect the expression level of GluN1 in hippocampal tissues, they did decrease the content of GluN1 on the cell membrane surface, which could be due to the antibodies promoting NMDAR internalization[22]. In this study, no apparent epileptic symptoms were observed in the mice. EEG recorded epileptic discharges in mice immunized with 78% GluN1_{359 − 378} peptides, including 6 positive mice and 1 negative mouse, suggesting clinical discharges in the anti-NMDA receptor encephalitis model immunized with GluN1_{359 − 378} peptides. The classical pathogenesis of abnormal discharge of neurons is that the imbalance of excitatory neurotransmitter glutamate and inhibitory neurotransmitter GABA leads to the imbalance of excitation and inhibition function of neurons[23]. Herein, western blot was utilized to detect the neurotransmitter vesicular glutamatetransporter (vGluT1) and γ-aminobutyric acid vesicle transporter (vGAT) levels. The former is a vesicular glutamate transporter whose function is to specifically transport glutamate outside synaptic vesicles into synaptic vesicles[24]. It was found that an increase of vGluT1 in hippocampal tissues immunized with GluN1359-378 peptides resulted in an increase in glutamate release, but the change in vGAT expression was not obvious, implying that GABA release was unaffected. Furthermore, our results highlighted that immunization with GluN1359-378 peptides could increase glutamate release and neuronal excitability. Our results corroborate those of Ganor et al., who used GluN1359-378 peptides to induce GluN3B antibodies against glutamate receptors in DBA mice, thereby lowering the epileptic threshold[14].

The mechanism of epilepsy caused by anti-NMDA receptor encephalitis is elusive. The present study demonstrated an increase in glutamate release in the anti-NMDA receptor encephalitis model immunized with GluN1_{359 − 378} peptides, which may be one of the key mechanisms underlying anti-NMDA receptor encephalitis seizure. Nevertheless, the receptor mechanisms related to neurotransmitters warrant further investigations.

In this study, GluN1_{359 − 378} was used as an immunogen to induce high titers of pathogenic anti-GluN1 autoantibodies, a convenient and effective substitute for an endogenous anti-NMDAR encephalitis model that is beneficial for studying the pathogenesis of NMDAR encephalitis.

The anti-NMDAR encephalitis model was successfully established by active immunization with GluN1_{359 − 378} peptide targeting GluN1 gene ATD, and the mouse EEG showed epileptiform discharge.

NMDAR: Anti-N-methyl-D-aspartate receptor

ATD: Amino terminal domain

NMDAR-AB: Anti-N-methyl-D-aspartate receptor antibody

EEG: Electroencephalogram

ELISA: Enzyme-linked immunosorbent assay

VGluT1: Glutamic vesicle transporters

vGAT: γ -Aminobutyric acid vesicle transporters

Ethics approval and consent to participate

All authors clearly consented to and approved this manuscript. The study strictly followed the Guiding Opinions on the Treatment of Laboratory Animals issued by the Ministry of Science and Technology of the People's Republic of China and the Laboratory Animal-Guideline for Ethical Review of Animal Welfare issued by the National Standard GB/T35892-2018 of the People's Republic of China. The study was approved by The Animal Care & Welfare Committee of Guangxi Medical University. The study strictly followed the ARRIVE guidelines.

Consent for publication

Not applicable.

Availability of Data and Materials

Not applicable.

Competing Interests

The authors have no competing interests to declare that are relevant to the content of this article.

Funding

This study was funded by National Natural Science Foundation of China (CN) (81760242). The funder had no role in the study design, data collection, or analysis, the decision to publish or the preparation of the manuscript.

Authors' Contributions

Study concept and design: HM H, Y W. Data collection: HM H, L Y. Analysis and interpretation: HM H, L Y, SJ L, XD L. Drafting of the manuscript: HM H. Critical revision of the manuscript: HM H, Y W. Study supervision: HM H, Y W. HM H was a major contributor in writing the manuscript. The authors read and approved the manuscript.

Acknowledgements

We are grateful to The Animal Care & Welfare Committee of Guangxi Medical University for the approval of our study.

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Construction of an active immune model and electrophysiological study of mice with anti-NMDAR encephalitis

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