ORIGINAL ARTICLE Neuropsychiatric disease relevance of circulating anti-NMDA receptor autoantibodies depends on blood–brain barrier integrity C Hammer 1 , B Stepniak 1 , A Schneider 2,3,4 , S Papiol 1,3 , M Tantra 1,3 , M Begemann 1 , A-L Sire ´n 5 , LA Pardo 6 , S Sperling 1 , S Mohd Jofrry 1 , A Gurvich 1 , N Jensen 1 , K Ostmeier 1 , F Lu ¨ hder 7 , C Probst 8 , H Martens 9 , M Gillis 10 , G Saher 11 , F Assogna 12 , G Spalletta 12 , W Sto ¨ cker 8 , TF Schulz 10 , K-A Nave 3,11 and H Ehrenreich 1,3 In 2007, a multifaceted syndrome, associated with anti-NMDA receptor autoantibodies (NMDAR-AB) of immunoglobulin-G isotype, has been described, which variably consists of psychosis, epilepsy, cognitive decline and extrapyramidal symptoms. Prevalence and significance of NMDAR-AB in complex neuropsychiatric disease versus health, however, have remained unclear. We tested sera of 2817 subjects (1325 healthy, 1081 schizophrenic, 263 Parkinson and 148 affective-disorder subjects) for presence of NMDAR-AB, conducted a genome-wide genetic association study, comparing AB carriers versus non-carriers, and assessed their influenza AB status. For mechanistic insight and documentation of AB functionality, in vivo experiments involving mice with deficient blood– brain barrier (ApoE  /  ) and in vitro endocytosis assays in primary cortical neurons were performed. In 10.5% of subjects, NMDAR- AB (NR1 subunit) of any immunoglobulin isotype were detected, with no difference in seroprevalence, titer or in vitro functionality between patients and healthy controls. Administration of extracted human serum to mice influenced basal and MK-801-induced activity in the open field only in ApoE  /  mice injected with NMDAR-AB-positive serum but not in respective controls. Seropositive schizophrenic patients with a history of neurotrauma or birth complications, indicating an at least temporarily compromised blood– brain barrier, had more neurological abnormalities than seronegative patients with comparable history. A common genetic variant (rs524991, P ¼ 6.15E  08) as well as past influenza A (P ¼ 0.024) or B (P ¼ 0.006) infection were identified as predisposing factors for NMDAR-AB seropositivity. The 410% overall seroprevalence of NMDAR-AB of both healthy individuals and patients is unexpectedly high. Clinical significance, however, apparently depends on association with past or present perturbations of blood–brain barrier function. Molecular Psychiatry advance online publication, 3 September 2013; doi:10.1038/mp.2013.110 Keywords: APOE; ApoE null mutant mice; autoimmunity; GWAS; influenza; NFIA INTRODUCTION N-methyl-D-aspartate receptors (NMDAR) are glutamate-gated ion channels, abundantly expressed in mammalian brain. 1 They form heteromers of NR1, NR2 and NR3 subunits, and are pivotal in regulating synapse function. 2 In schizophrenia, NMDAR hypofunction has been hypothesized due to induction of psychotic symptoms by antagonists. 3 In 2007, Dalmau et al. 4,5 described a paraneoplastic syndrome, based on 12 women with ovarian teratoma, carrying IgG autoantibodies (AB) against the NMDAR NR1/2 subunits. The syndrome, termed ’anti-NMDAR encephalitis’, variably consisted of psychosis, memory deficits, seizures, dyskinesia, decreased consciousness and autonomic instability. Since its initial description, a flood of publications appeared. The search for anti-NR1 IgG AB in small samples (N ¼ 46–80) of schizophrenic patients yielded discordant results. 6–8 Recently, 4400 previously collected cases of anti-NMDAR encephalitis have been reviewed, most without associated tumor. 9 Similarly, immunomodulatory treatment outcomes of these and around 100 more cases have been summarized. 10 As a syndrome- pertinent pathophysiological mechanism, an AB-induced decrease of NMDAR-mediated currents, due to enhanced receptor internalization and thus reduced surface expression, has been suggested. 11 Few studies explored a role of other classes of immunoglobulins (Ig) in an NMDAR-AB syndrome. In individuals with slow cognitive impairment, anti-NR1 IgA AB were found, which affected synaptic protein expression and decreased NMDAR-mediated currents. 12 Anti-NR1 IgM AB were described in a patient with bipolar disorder 13 and in patients with herpes simplex encephalitis. 14 In the largest study so far, investigating IgG, IgA and IgM, Steiner et al. 15 reported a higher prevalence of AB of all isotypes in 121 schizophrenic patients, compared with healthy controls or patients suffering from affective disorders. Apart from tumors, no sound information is available yet on putative susceptibility factors for the development of anti-NR1 AB. The present study was designed to (1) systematically screen in an unbiased fashion a large number (N ¼ 2817) of healthy 1 Clinical Neuroscience, Max Planck Institute of Experimental Medicine, Go ¨ ttingen, Germany; 2 Department of Psychiatry & Psychotherapy, University Medicine Go ¨ ttingen, Go ¨ ttingen, Germany; 3 DFG Research Center Nanoscale Microscopy and Molecular Physiology of the Brain (CNMPB), Go ¨ ttingen, Germany; 4 German Center for Neurodegenerative Diseases (DZNE), Go ¨ ttingen, Germany; 5 Department of Neurosurgery, University Clinic of Wu ¨ rzburg, Wu ¨ rzburg, Germany; 6 Department of Molecular Biology of Neuronal Signals, Max Planck Institute of Experimental Medicine, Go ¨ ttingen, Germany; 7 Department of Neuroimmunology, Institute for Multiple Sclerosis Research and Hertie Foundation, University Medicine Go ¨ ttingen, Go ¨ ttingen, Germany; 8 Institute for Experimental Immunology, affiliated to Euroimmun, Lu ¨ beck, Germany; 9 Synaptic Systems GmbH, Go ¨ ttingen, Germany; 10 Institute of Virology, Hannover Medical School, Hannover, Germany; 11 Department of Neurogenetics, Max Planck Institute of Experimental Medicine, Go ¨ ttingen, Germany and 12 Department of Clinical and Behavioral Neurology, IRCCS Santa Lucia Foundation, Rome, Italy. Correspondence: Professor H Ehrenreich, Clinical Neuroscience, Max Planck Institute of Experimental Medicine, Go ¨ ttingen 37075, Germany. E-mail: ehrenreich@em.mpg.de Received 22 May 2013; revised 19 July 2013; accepted 22 July 2013 Molecular Psychiatry (2013), 1–7 & 2013 Macmillan Publishers Limited All rights reserved 1359-4184/13 www.nature.com/mp