Anti-N-methyl-D-aspartate receptor (NMDA) receptor encephalitis is a recently discovered autoimmune syndrome associated with psychosis, dyskinesias, and seizures. Ectopic expression of NMDA receptors associated with ovarian teratoma is thought to mediate the initial autoimmune response against NMDA receptor encephalitis. Due to the lack of suitable animal models, the underlying mechanism of the disease remains unclear. This study described a new mice model of active immunization against the

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... receptor with amino-terminal domain (ATD) peptides. After 12 weeks of immunization, mice were showed significant behavioral disorders and memory loss. Antibodies from CSF of immunized mice decreased surface NMDAR cluster density on hippocampus neurons. It also impaired the LTP induced at the Schaffer collateral to CA1 synapse and reduced NMDA receptors-induced calcium influx. The new model may help further research into the pathogenesis of the disease and the development of potential new therapies. Introduction Anti-N-methyl-D-aspartate receptor (NMDA) receptor encephalitis predominantly affects children and young women. Almost 80% of patients showed abnormal in cerebral spinal fluid (CSF), presenting as mildly lymphocytic pleocytosis, normal or moderate increased protein levels, and most importantly, specific oligoclonal bands appear in 60% of patients [25, 29]. Ectopic expression of NMDA receptor is thought to trigger the immune response in anti-NMDA receptor encephalitis [1, 27]. Ovarian teratomas and other tumors have been shown to express the NMDA receptor, potentially explaining the association [5]. Antigens released by tumor cells are taken up by antigen-presenting cells that travel to regional lymph nodes, plasma cells produce antibodies that later cross-react with NMDA receptor in the brain following impaired blood-brain barrier permeability [7, 13, 21] . NMDA receptor mediate glutamatergic synaptic transmission and have a prominent role in synaptic plasticity [4, 15] . Previous studies have suggested that the pathogenicity of autoimmune antibodies (ABs) is one of the key mechanisms of anti-NMDA receptor encephalitis [14, 17, 20] . Human CSF-derived NMDA receptor ABs downregulates the level of NMDA receptor both in in vitro and in vivo studies [22] . Meanwhile, the reduced expression of NMDA receptors can result in the increase in extracellular glutamate and further affect the pons medullary respiratory center [22] . The passive immunized mice develop symptoms of depression, anhedonia, and memory deficits following the intrathecal infusion of CSF from affected humans, it provides evidence of the effect of autoantibodies on the disease, but do not produce the pathogenesis of anti-NMDA receptor encephalitis in human [1, 2]. To better mimic the clinical manifestations and mechanism of anti-NMDA receptor encephalitis, it is still worth developing an animal model of active immunity that mimics disease progression. Previous investigations highlighted the extracellular amino-terminal domain (ATD) of the GluN1 subunit, especially the N368/G369 region that is essential for immunoreactivity [11, 23, 26]. and ATD peptides were used to immunize and induce fulminant encephalitis in mice within 12 weeks. The mice demonstrated behavioral changes and ABs infiltration that were most prominent in the hippocampus. The presence of NMDA receptor ABs was confirmed by and its effect on the NMDA receptor was also confirmed. Materials And Methods Study design and mice immunization The study aimed to investigate the effect of active immunization with NMDA receptor peptides on normal adult mice. C57BL/6 Mice (10 weeks old) were immunized with different GluN1 extracellular peptides and emulsified in an equal volume of complete Freund's adjuvant supplemented with of Mycobacterium tuberculosis H37Ra (4 mg/mL) at a final concentration of 1 mg/mL. At the tail base, 200 μg of GluN1 peptides or control peptides were injected subcutaneously and boosted twice at 4 and 8 weeks after the first immunization, respectively. All mice were intraperitoneally injected with 400 ng pertussis toxin (PTX) at the last immunization. Behavioral tests, histological staining analysis were performed 10 weeks after the first immunization. Patients' sample collection We collected CSF from patients with high titer of anti-GluN1 ABs (>1:300) during routine clinical practice. All patients fulfilled the clinical diagnostic criteria for anti-NMDAR encephalitis revised in 2016 [12]. The study protocol was approved by the ethics committee of the Nanfang Hospital, 5 Southern Medical University, and written informed consent was obtained from each participant. Antibody purification CSF and serum from patients or immunized mice were purified with protein G Sepharose columns and used to treat neurons or brain slices. 2 ml of diluted sample was incubated with a chromatography spin column (Thermo Scientific) of protein G Sepharose beads for 30 min. After three washes with PBS, the sample were eluted with elution buffer, dialyzed against PBS, concentrated in stock solutions of 4 mg/mL and stored at -80 °C until used. Preparation and staining of GluN1-expressing HEK cells Human embryonic kidney 293 (HEK) cells were transiently transfected with NMDA receptor subunit genes (NR1/NR2A) (DsRed2 labeled) as described [24] . 24 hours later, cells on coverslips were fixed with acetone and incubated with the CSF from patients or immunized mice (starting at 1:1) in 0.1% bovine serum albumin (BSA) in PBS overnight at 4 °C. After washed with PBS, cells were labeled with FITC-conjugated anti-human IgG and observed under a fluorescence microscope (BX51, Olympus) Site-directed mutagenesis Point mutation were made using the Stratagene QuikChange Mutagenesis kit (210518, Agilent) according to the manufacturer's instruction. Primers designed for N368Q point mutation: Forward: 5'gggatgacatgggtaccttggtagatgcccacttgca-3'; Reverse:5'-tgcaagtgggcatctaccaaggtacccat gtcatccc-3'. Primary neuron culture Hippocampal neurons were prepared and maintained from embryonic day 18 rat brains. The hippocampi were dissociated with papain at 37 °C for 30 minutes, and then separated with a fire polished Pasteur pipette. After centrifugation at 300 g, the cells were resuspended in neurobasal medium. Cells were counted and plated on poly-D-lysine (PDL) treated 24-well plates. After 6 hours, the supernatant was removed and replaced with 500 μL of fresh culture medium. Cells were cultured for 14 days for subsequent experiments. Immunostaining Immunostaining is used to detect binding to brain slices and autoantibodies. On day 14 after immunization, CSF was obtained from mice, purified and incubated with brain slice. Slices were Not applicable. (2017) All naturally occurring autoantibodies against the NMDA receptor subunit NR1 have pathogenic potential irrespective of epitope and immunoglobulin class. Mol teratoma associated with anti-N-methyl D-aspartate receptor encephalitis: a report of 5 cases documenting prominent intratumoral lymphoid infiltrates. Int J Gynecol Pathol 31: 429-437 Doi 10.1097/PGP.0b013e31824a1de2 6 Dalmau J (2016) NMDA receptor encephalitis and other antibody-mediated disorders of the synapse: The 2016 Cotzias Lecture.