{'fraction_non_alphanumeric': 0.0500747703482162, 'fraction_numerical': 0.02324289681691946, 'mean_word_length': 5.05588615782665, 'pattern_counts': {'":': 0, '<': 4, '<?xml version=': 0, '>': 9, 'https://': 0, 'lorem ipsum': 0, 'www.': 0, 'xml': 0}, 'pii_count': 1, 'substrings_counts': 0, 'word_list_counts': {'cursed_substrings.json': 0, 'profanity_word_list.json': 0, 'sexual_word_list.json': 0, 'zh_pornsignals.json': 0}}
Uncoupling the widespread occurrence of anti-NMDAR1 autoantibodies from neuropsychiatric disease in a novel autoimmune model

Autoantibodies of the IgG class against N-methyl-D-aspartate-receptor subunit-NR1 (NMDAR1-AB) were considered pathognomonic for anti-NMDAR encephalitis. This view has been challenged by the age-dependent seroprevalence (up to >20%) of functional NMDAR1-AB of all immunoglobulin classes found in >5000 individuals, healthy or affected by different diseases. These findings question a merely encephalitogenic role of NMDAR1-AB. Here, we show that NMDAR1-AB belong to the normal autoimmune repertoire of dogs, cats, rats, mice, baboons, and rhesus macaques, and are functional in the NMDAR1 internalization assay based on human IPSC-derived cortical neurons. The age dependence of seroprevalence is lost in nonhuman primates in captivity and in human migrants, raising the intriguing possibility that chronic life stress may be related to NMDAR1-AB formation, predominantly of the IgA class. Active immunization of ApoE−/− and ApoE+/+ mice against four peptides of the extracellular NMDAR1 domain or ovalbumin (control) leads to high circulating levels of specific AB. After 4 weeks, the endogenously formed NMDAR1-AB (IgG) induce psychosis-like symptoms upon MK-801 challenge in ApoE−/− mice, characterized by an open blood–brain barrier, but not in their ApoE+/+ littermates, which are indistinguishable from ovalbumin controls. Importantly, NMDAR1-AB do not induce any sign of inflammation in the brain. Immunohistochemical staining for microglial activation markers and T lymphocytes in the hippocampus yields comparable results in ApoE−/− and ApoE+/+ mice, irrespective of immunization against NMDAR1 or ovalbumin. These data suggest that NMDAR1-AB of the IgG class shape behavioral phenotypes upon access to the brain but do not cause brain inflammation on their own.

* Hannelore Ehrenreich ehrenreich@em.mpg.de Extended author information available on the last page of the article MOG, and CASPR2. In contrast, AB that recognize intracellular antigens, e.g., amphiphysin, ARHGAP26, or GAD65, show predominance of IgG [6]. Despite this apparent "physiological autoimmunity", no report exists that systematically screened mammals other than humans for the presence of NMDAR1-AB. In recent work, we found that all naturally occurring NMDAR1-AB are functional and thus have pathogenic potential irrespective of epitope and Ig class [10]. Pathophysiological significance may emerge in conditions of compromised blood-brain barrier (BBB), for instance, upon injury, infection, inflammation, or genetic predisposition (APOE4 haplotype), which then allows substantial access of circulating NMDAR1-AB to the brain where they act as NMDAR antagonists [5,9,[18][19][20]. Alternatively, AB-specific plasma cells may reside or settle in the brain and produce large amounts of AB intrathecally [14,21]. The question whether abundant endogenously produced NMDAR1-AB of the IgG class can-upon access to the brain-induce inflammation and thus "anti-NMDAR1 encephalitis" has never been experimentally addressed.
The present paper has therefore been designed to (i) systematically screen mammals other than humans for seroprevalence of functional NMDAR1-AB and (ii) study mice with open BBB behavioral and morphological consequences of high circulating levels of endogenous NMDAR1-AB of the IgG class formed in response to immunization.

Ethical approvals
Ethics committees of Georg-August University, Göttingen, and collaborating centers approved the Göttingen Research Association for Schizophrenia (GRAS) data collection and other studies "extended GRAS" acquiring human data, serum samples, and IPSC [5,6,8,9,22,23]. Hannover Medical School Ethics Committee approved the neurosurgical specimen collection. Studies comply with Helsinki Declaration. Patients gave written informed consent. Mouse studies were approved by Animal Ethics (LAVES, Oldenburg) following German Animal Protection Law.
Notes: All experiments were performed by researchers unaware of group assignment. The new nomenclature GluN1 for NMDAR1 is mostly disregarded here for consistency with the respective literature.

Dogs and cats
Serum samples from dogs and cats of different breeds were prospectively collected during routine (health check/vaccination) or diagnostic (spectrum of different disorders) workup of outpatients in the Small Animal Clinic, University of Veterinary Medicine, Hannover.

Monkeys
Serum samples from healthy baboons and rhesus macaques were obtained through routine checkups at the Leibniz Institute for Primate Research, Göttingen.

Rodents
Serum samples from healthy rats and mice were obtained at the Max Planck Institute of Experimental Medicine and the Institute for Multiple Sclerosis Research, Göttingen.

NMDAR1-AB determination by clinical standard procedures
Human serum and ventricular CSF were tested for NMDAR1-AB positivity using commercially available kits, based on HEK293T cells transfected with NMDAR1 and secondary AB against human IgG, IgM, or IgA (Euroimmun, Lübeck, Germany) [2,25]. Mouse serum was  analyzed using the same assay with secondary AB against  mouse IgG, IgM, or IgA (M31001, A-31570, A-21042;  Thermo Fisher, Rockford, USA).

Protein-A assay
Human serum (for cross-validating clinical standard procedure and protein-A method), as well as dog, cat, rat, and monkey serum were labeled with protein-A from Staphylococcus aureus, binding the Fc portion of immunoglobulins of different species [26]. Plasma (50 μl) and 25 μg of FITC-conjugated protein-A (Sigma-Aldrich) were incubated for 2 h in the dark at room temperature (RT). The mixture was then diluted to 250 μl (PBS) and unbound FITC-Protein-A was removed using 100-kDa Amicon filter units (Sartorius, Göttingen, Germany), reconcentrating tõ 50 μl [27]. NMDAR1-AB seropositivity was determined using Euroimmun assay combined with commercial monoclonal mouse NMDAR1-AB (114011; M68, SYSY, Göttingen, Germany). Samples showing distinct double labeling were rated "positive" (>98% consensus of three investigators).

BBB-integrity testing
BBB integrity of 12-month-old ApoE −/− (N = 5) and ApoE +/+ (N = 5) mice was determined using two different fluorescent tracers, Evans blue (50 mg/g body weight) [30] and sodium fluorescein (200 mg/g body weight). A detailed description of this method will be published elsewhere [31]. Briefly, for tracer quantification in the brain at 4 h after intravenous injection in the tail vein, animals were PBS perfused to remove the circulating tracer. Brains were dissected, immediately frozen on dry ice, weighed, and stored at −80°C. Tissue was lyophilized at −56°C for 24 h under vacuum of 0.2 mBar (Christ LMC-1-BETA-1-16, Osterode, Germany). For tracer extraction, hemispheres were incubated with shaking in 10 ml formamide/mg brain at 57°C for 24 h. Integrated density of tracer fluorescence was determined in triplicates on a fluorescent microscope (Observer Z2, Zeiss, Germany), equipped with Axio-CamMRc3, 1×Camera-Adapter, and ZEN2012 blue-edition software, recorded at 10× magnification (Plan-Apochromat 10×/0.45M27). Tracer concentration was calculated using a standard curve and normalized to controls (set to 1).

Statistical analyses
Statistical analyses were performed using SPSSv.17 (IBM-Deutschland-GmbH, Munich, Germany) or Prism4 (GraphPad Software, San Diego, California, USA). Group differences in categorical and continuous variables were assessed using χ 2 , Mann-Whitney U, or Student's t-tests depending on data distribution/variance homogeneity. ANOVA was employed as indicated in display item legends. All p-values are two tailed; significance is set to p < 0.05; data are presented as mean ± S.E.M.

Cross-validation of NMDAR1-AB detection methods
To determine NMDAR1-AB seropositivity in mammals other than humans, we had to validate the protein-A detection method [27]. For that, N = 72 paired human serum and ventricular CSF samples, prospectively collected from random neurosurgical patients, were analyzed by the usual cell-based assay, employing specific secondary AB for all Ig classes. A total of N = 5 sera turned out NMDAR1-AB positive (titers ≤ 1:100; 3× IgM; 2× IgA; 0× IgG). Ventricular CSF samples were all negative. For cross-validation of NMDAR1-AB of the IgG class, we used serum of a seropositive stroke patient [8]. Application of protein-A method combined with double labeling for NMDAR1-AB M68 confirmed positive and negative results (Fig. 1a).

High seroprevalence of NMDAR1-AB across mammalian species
We next analyzed by protein-A method serum samples of dogs, cats, rats, baboons, and rhesus macaques. Strikingly, all mammalian species, independent of their respective life expectancy, show high NMDAR1-AB seropositivity (Fig. 1b). Mouse samples were analyzed using specific AB against murine IgA, IgM, and IgG. As known for humans [6], NMDAR1-AB of the IgG class were the rarest. For another cross-validation, all monkey samples (N = 100) were analyzed in blinded fashion by an independent lab (Bochum; using specific anti-monkey IgM). IgM-positive results coincided with the protein-A positivity by >97% (76 of 78). The fraction of protein-A positive but IgMnegative monkey samples (total 22%) likely presents NMDAR1-AB of IgA class and IgG class where specific AB were not available.
Age-dependent NMDAR1-AB seroprevalence except for nonhuman primates and human migrants All species revealed age dependence of NMDAR1-AB seroprevalence (χ 2 test; dogs: χ 2 (1) = 11.5, p = 0.01; cats: χ 2 (1) = 4.8, p = 0.03; rats: χ 2 (1) = 9.5, p = 0.002; and mice: Fisher's exact test p = 0.032) as for humans [5,8] with the exception of baboons (χ 2 (1) = 1.0, p = 0.3), where already >50% of young animals were seropositive. This surprising result made us investigate another monkey species, rhesus macaques, showing again high seroprevalence in old and young animals (χ 2 (1) = 0.2, p = 0.6) (Fig. 1b). We wondered what the difference between humans, dogs, cats, mice, and rats, on one hand, and monkeys, on the other hand, could be, leading to loss of the usual age pattern regarding seroprevalence. Postulating that captivity/nondomestication of young monkeys might induce chronic life stress due to maladaptation to the environment, we investigated in a hypothesis-driven way whether young human migrants would display a similar increase in NMDAR1-AB seropositivity. Of the GRAS data collection, detailed information on migration was available in a subsample of N = 970 individuals. While nonmigrants show the typical age association of NMDAR1-AB seroprevalence (χ 2 (1) = 10.7, p = 0.001), migrants do not (χ 2 (1) = 0.6, p = 0.4) (Fig. 1c). Seroprevalence in young migrants is significantly higher as compared to young nonmigrants (χ 2 (1) = 5.381, p = 0.020). In both monkey species and migrants, the IgM fraction still follows the expected age trend, while IgA seems to account for the early increase in NMDAR1-AB seroprevalence (Fig. 1c). Presentation of NMDAR1-AB by Ig class in the extended GRAS sample (N = 4933), with N = 4632 of likely nonmigrants (available information less detailed) and N = 301 known migrants, illustrates the abnormal course of IgA vs. IgM/IgG seroprevalence over age in migrants (Fig. 1d).

Functionality of NMDAR1-AB from different mammalian species
To assess whether NMDAR1-AB of the tested species are functional, our endocytosis assay using IPSC-derived human cortical neurons [10] was employed. All positive sera provoked NMDAR1 internalization, verifying functionality (Mann-Whitney U; all p < 0.001) (Fig. 1e). Step 1

BBB dysfunction but normal behavior of
Step 2  a Cross-validation of assays: paired serum and intraventricular CSF samples from neurosurgical patients were tested using a HEK293T cell-based clinical standard assay for NMDAR1-AB seropositivity (Euroimmun biochip). For step 1, fluorescently labeled IgA-specific, IgM-specific, and IgG-specific secondary AB were used; for method cross-validation (step 2), NMDAR1-AB seropositive and seronegative samples of each Ig class from step 1 were labeled with protein-A-FITC conjugate and tested for colocalization (yellow) of protein-A-FITC + (green) and M68 + (monoclonal mouse NMDAR1-AB followed by Alexa555 donkey anti-mouse IgG red). Representative pictures of both methods using the same seropositive samples (IgA, IgM, and IgG) are displayed on the right: upper row step 1/lower row step 2. b NMDAR-AB seropositivity (%) of young and old mammals for all Ig classes combined ( # protein-A-FITC/Euroimmun) or for individual classes ( + Euroimmun; *protein-A-FITC/Euroimmun and cross-validation with Euroimmun/monkey IgM) presented in the bars; color codes used for consistency and kept also in c and d; age given in months (m) or years (y); χ 2 or Fisher's exact test. c NMDAR-AB seropositivity of subjects with migration (first and second generation) vs. nonmigration history (GRAS data collection); all Ig classes presented; age split at 35 years; χ 2 test. d NMDAR1-AB course by Ig classes in serum over age groups in migrants vs. nonmigrants of the extended GRAS data collection. Note the different course particularly for IgA. eFunctionality testing of NMDAR1-AB in human IPSCderived cortical neurons: degree of internalization expressed as a ratio of fluorescence intensity measured at 37 and 4°C; number of neurons and sera (N) given; Mann-Whitney U test

GluN1 immunizaƟon (IgG)
GluN2B e Quantification of Iba1 + and CD3 + cells in the hippocampus to assess inflammation in the brain; one-way ANOVA; representative pictures of Iba1 (left) and CD3 (right) stainings in the middle month-old mice (age of immunization) BBB leakiness using two fluorescent tracers. While brain water content was similar in both genotypes, pointing against inflammation, ApoE −/− mice showed increased tracer extravasation, confirming BBB dysfunction (Student's t-test: EB: t(8) = −10.66, p < 0.001; NaFl: t(8) = −8.97, p < 0.001) (Fig. 2a). We wondered whether this compromised BBB would by itself lead to behavioral abnormalities in ApoE −/− mice. A comprehensive behavioral battery, including tests for anxiety, activity, exploratory behavior, motor and sensory function, sensorimotor gating, pheromone-based social preference, and cognitive performance did not reveal any differences between genotypes ( Table 1).
Immunization of ApoE −/− and ApoE +/+ mice against NMDAR1-peptides To explore whether endogenously formed NMDAR1-AB would lead to measurable behavioral and morphological effects, we immunized 12-month-old ApoE −/− and ApoE +/+ littermates against four peptides of the extracellular NMDAR1/GluN1-domain (including NTD-G7; N368/ G369) and ovalbumin or against ovalbumin alone as immunization control (Fig. 2b-c). GluN1 shows >99% sequence homology among all here-tested mammalian species, with immunizing peptides being 100% homologous (Fig. 3). Immunization led to high circulating levels of specific IgG (titers up to 1:50,000). Efficacy of immunization and time course of IgG appearance as determined by ELISA were comparable for NMDAR1-peptides and ovalbumin across genotypes, making a simple boosting effect of NMDAR1-peptides on pre-existing NMDAR1-specific B cell clones rather improbable (Fig. 2c).

Psychosis-related behavior of ApoE −/− mice upon MK-801 challenge
Open-field tests measuring baseline preimmunization and postimmunization locomotion did not reveal any differences between genotypes and/or immunization groups ( Fig. 2b; not shown). After 4 weeks, the endogenously formed NMDAR1-AB of the IgG class induced strong hyperactivity (psychosis-like symptoms [37]) upon MK-801 challenge in ApoE −/− mice only. In contrast, ApoE +/+ mice behaved comparably to ovalbumin-only immunized mice of both genotypes ( Fig. 2d; all p > 0.5). Thus, an open BBB together with sufficiently high titers of AB (to reach a threshold loss of NMDAR1 surface expression) is a prerequisite for the observed behavioral perturbation upon MK-801.
No inflammation in hippocampus of immunized ApoE −/− and ApoE +/+ mice Immunohistochemistry did not show any evidence of inflammation in either genotype and/or immunization group. Numbers of Iba1 + and CD3 + cells as markers of microglia and T cells, respectively, were comparable for total hippocampus (one-way ANOVA: Iba1: F(3,18) = 0.3; p = 0.8; CD3: F(3,18) = 0.4; p = 0.8) (Fig. 2e) and for all hippocampal subfields separately (all p-values > 0.2; not shown). Also, staining for microglial activity markers, CD68 and MHCII, was essentially negative and identical across groups. Moreover, staining for GFAP did not reveal any appreciable density increase in the hippocampus, and thus no sign of astrogliosis (data not shown).

Discussion
The present work demonstrates high seroprevalence of functional NMDAR1-AB of all Ig classes across mammals, indicating that these AB are part of a pre-existing autoimmune repertoire [11][12][13][14][15][16][17]. As in humans, NMDAR1-AB of the IgG class are the least frequent [6,20]. The age related up to >50% NMDAR1-AB seropositivity is independent of the respective species' life expectancy, indicating that the aging process itself rather than years of exposure to a certain environment triggers NMDAR1-AB formation. However, our knowledge on predisposing factors and inducing mechanisms is limited. Specific autoimmunereactive B cells may get repeatedly boosted by, e.g., infections, neoplasms, or the microbiome, and less efficiently suppressed over a lifespan likely owing to a gradual loss of immune tolerance upon aging [14]. Unexpectedly, we find the age-dependence lost in nonhuman primates and in human migrants that all display an early-life rise in NMDAR1-AB seropositivity, mainly of IgA. The intriguing possibility that chronic life stress, known to be present in human migrants [38] and animals in captivity [39], acts as a trigger of early NMDAR1-AB formation is worth pursuing experimentally in the future. A large proportion of migrants in our human samples are suffering from neuropsychiatric illness. This may additionally support our chronic stress hypothesis since migration is recognized as an environmental stressor predisposing to mental disease [40]. Further studies should screen wildlife monkeys and species in zoos for NMDAR1-AB. Experimental confirmation of our findings provided, NMDAR1-AB (IgA) may even serve as stress markers. In fact, earlier reports show that total serum-Ig of all classes, most prominently IgA, respond to psychological stress [41]. NMDAR1-AB might thus belong to a set of stress-boosted AB. Interestingly, we also find accumulated seroprevalence of 23 other brain-directed AB [6] in young migrants vs. nonmigrants increased (data not shown), suggesting a global inducer role of chronic stress in humoral autoimmunity.
Earlier work has shown that AB against brain antigens in general are common among mammals [42], but no study has so far systematically screened nonhuman mammals for NMDAR1-AB. As an exception, a recent report described "anti-NMDAR1 encephalitis" in the young polar bear Knut [27]. Based on the present findings, Knut may have belonged to those nondomesticated species in captivity-  Fig. 2c) and nonhomologous amino acids in pink. SP signal peptide, S1, S2 segments of the ligand-binding domain, TMD A transmembrane domain A, TMD B transmembrane domain B, TMD C transmembrane domain C comparable to monkey species investigated here-that are affected by chronic early-life stress, inducing NMDAR1-AB seropositivity. Pre-existing NMDAR1-AB of this bear may have ultimately shaped the clinical picture of an encephalitis of unexplained origin (likely infectious according to the zoo's pathology reports) where an epileptic seizure led to drowning [27]. This interpretation is supported by our novel autoimmune model, namely, mice immunized against NMDAR1-peptides. Even high titers of endogenously formed NMDAR1-AB (IgG; up to 1:50,000) that induce psychosis-like behavior upon MK-801 challenge in ApoE −/− mice, with here-confirmed open BBB, do not lead to any appreciable signs of encephalitis. This dissociation of behavioral/symptomatic consequences and inflammation in the brain is of major importance for clinicians [14]. For instance, earlier studies reported an influence of NMDAR1-AB infusions into the hippocampus on learning and memory in mice [43], and others found increased NMDAR1-AB seroprevalence in patients with mild cognitive impairment and Alzheimer's disease [44,45]. However, while all naturally occurring NMDAR1-AB that have pathogenic potential irrespective of epitope and Ig class [10], and upon entry to the brain (or via intrathecal production) can shape brain functions in the sense of NMDAR antagonism, only a fraction of individuals happens to have underlying encephalitis of various etiologies, which is then called anti-NMDAR encephalitis. The highly variable neuropathology and response to immunosuppression of this condition [2,3,46] may point to a broad range of possible encephalitogenic mechanisms (from infection to oncology or genetics) which need to be diagnosed and specifically treated [14].
Even though it is unclear how NMDAR1-AB are generated by chronic stress, it should be considered that NMDAR1 are not only expressed in the brain but also by peripheral organs and tissues, including adrenal glands and gut [47] which may be involved in triggering NMDAR1-AB formation but may also be functionally modulated by them. Since NMDAR antagonists are increasingly recognized as antidepressant, anxiolytic, and anti-inflammatory agents [48][49][50][51][52], we speculate that stress-induced NMDAR1-AB could serve as endogenous stress protectants. Remarkably, also in stroke, NMDAR1-AB can be protective [8].
In conclusion, the widespread occurrence of NMDAR1-AB across mammals, as well as the failure of even high titers of endogenously formed NMDAR1-AB of the IgG class to induce any signs of brain inflammation should lead to rethinking current concepts that link NMDAR1-AB to neuropsychiatric disease including encephalitis.

Compliance with ethical standards
Conflict of interest WS is a member of the board and holds stocks in Euroimmun AG. HM is a full-time employee of Synaptic Systems GmbH. The remaining authors declare that they have no conflict of interest.
Open Access This article is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License, which permits any non-commercial use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. If you remix, transform, or build upon this article or a part thereof, you must distribute your contributions under the same license as the original. The images or other third party material in this article are included in the article's Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons. org/licenses/by-nc-sa/4.0/.