Elevated serum anti-NMDA receptor antibody levels in first-episode patients with schizophrenia
Introduction
Schizophrenia is a complex and heterogeneous mental disorder. Although no unifying pathophysiologic abnormalities have been identified, neurodevelopmental, neuroimmunological, and neurotransmitter-based hypotheses have been proposed for the pathogenesis of the illness (Harrison and Weinberger, 2005). Accumulating evidence has demonstrated that schizophrenia is associated with immunological findings in the blood or cerebrospinal fluid (CSF) (Steiner et al., 2013), and that patients with schizophrenia exhibit increases in glial cell count and blood-brain/blood-CSF barrier dysfunction (Maxeiner et al., 2009, Bechter et al., 2010, Busse et al., 2012).
NMDA-type glutamate receptors are ligand-gated ion channels that mediate a major component of excitatory neurotransmission in the central nervous system (CNS). They are widely distributed in the CNS at all stages of development and are critically involved in neuronal development and synaptic plasticity. In the course of brain development, if the NMDA receptors receive proper stimulation, the neurons will survive. However, if the NMDA receptors are removed, apoptosis will be induced within a few hours (Ikonomidou et al., 1999). Under physiological conditions, the stimulation of NMDA receptors represents an important mechanism for synaptic pruning during brain development (Contestabile, 2000). The hypofunction of NMDA receptors has also been associated with multiple neurological and psychiatric disorders, such as ischemic stroke, traumatic brain injury, Alzheimer's disease, epilepsy, mood disorders, and schizophrenia (Hansen et al., 2017, Warikoo et al., 2018).
The NMDAR is an isotetramer that consists of three subunits: NR1, NR2, and NR3. NR1 contains eight different subunits (NR1-1a/b-4a/b); NR2 contains four different subunits (NR2A-2D); and NR3 contains two different subunits (NR3A-3B). While all eight subunits of NR1 are derived from the same gene yet generated from different parts of the shear; six different genes code NR2 and NR3. In mammalian nervous tissues, the functional NMDAR is composed of at least one NR1 and two NR2 subunits; but most NMDARs consist of two NR1 subunits and two NR2 subunits in the two-polymer combination. NR1 is the basic subunit of ion channels, while NR2 is a regulatory subunit. NMDARs consist of different types of NR2, which are distributed in different brain regions and give rise to different physiological characteristics (Gleichman et al., 2012). Convergent evidence indicates that NMDAR signaling is impaired in schizophrenia (Emamian et al., 2004, Ayalew et al., 2012, Weickert et al., 2013, Ohi et al., 2015), although the mechanism underlying the NMDAR hypofunction in patients with schizophrenia remains unclear. Growing evidence suggests that anti-NMDAR Abs is involved in schizophrenia.
However, studies on this have shown inconsistent results. Rhoads et al. and Masdeu et al. observed no anti-NMDAR autoantibodies in the serum of patients with schizophrenia (Rhoads et al., 2011, Masdeu et al., 2012), while Zandi et al. observed NMDAR serum antibodies in approximately 6% (3/46 patients) with first-episode schizophrenia (Zandi et al., 2011). In another study, immunoglobulin G (IgG)-class antibodies directed against NR1a were found in only two patients with an initial diagnosis of disorganized or catatonic schizophrenia, both of whom were subsequently rediagnosed with NMDAR encephalitis. In contrast, two other individuals with IgG antibodies received diagnoses of paranoid schizophrenia, and the antibodies did not bind to NR1a but were only reactive with NR1a/NR2b (Steiner et al., 2013). Lennox et al found 7 of 228 (3%) patients had NMDAR antibodies in first-episode psychosis (Lennox et al., 2017). Autoantibodies of the IgG isotype against NMDAR were also detected by Tsutsui et al. in 4 of 51 (8%) patients with schizophrenia (Tsutsui et al., 2012). In a review involving seven studies comprising 1441 patients with schizophrenia and related psychoses, among whom 115 were positive for anti-NMDAR Abs (Pollak et al., 2014).
As we know, immunofluorescence to detect NMDAR antibodies by their binding to the surface of HEK293A cells functionally expressing NMDAR was common and reliable detection method until recently (Ramberger et al., 2015). However, it is complex and difficult to implement and yields qualitative rather than quantitative results. NR1 is the basic subunit of ion channels and the functional subunit of NMDAR. Further, the amino terminal domain (ATD) of GluN1 is required for binding of anti-NMDAR Abs (Dalmau et al., 2008), and residues N368/G369 expressed in the N-terminal domain are crucial for antibody binding (Gleichman et al., 2012). The terminal sequence of NMDAR was known to be mature for the construction of polypeptide. The sequences were QKRLETLLEERESK (AA177-190) for the N-terminal of GluN1 (GluN1-NT), and SS FKRRRSSKDTST (AA889-902) for the C-terminal of GluN1 (GluN1-CT) (Fujita et al., 2012, Fukuyama et al., 2015, Ikura et al., 2016). And Sharma et al. (2018) found the ATD fusion protein also contains a Myc tag and a 6XHIS tag, which provide functionality for immunoassays and soluble antigen formats maintain the pathogenic anti-N-methyl-D-aspartate receptor encephalitis (ANRE) epitopes in ELISA binding assays, which makes quantitative research become reality. In recent years, ELISA has been continuously used to detect NMDAR antibody levels (Kowal and Diamond, 2012, Kalev-Zylinska et al., 2013, Ogawa et al., 2016, Ferensztajn-Rochowiak et al., 2019).
To our knowledge, there is still no published study that has examined the relationship between NMDAR antibody levels and clinical features in first-episode antipsychotics-naïve patients. Studies have shown that the blood-brain barrier is damaged in patients with schizophrenia, thus increasing the number of Abs in the blood. Furthermore, one study (Castillo-Gomez et al., 2016) has demonstrated that Abs have crossed the blood-brain barrier to bind to the brain - with the brain acting as an immunoprecipitator – and are therefore unmeasurable in the CSF. Therefore, serum antibody levels may be a better option to reflect the levels of NMDAR Abs in brain. Therefore, in this study, we evaluated the presence of serum Abs directed against the NR1 subunit with ELISA.
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Participants
One hundred and ten first-episode patients with schizophrenia were recruited for this study from Beijing Huilongguan Hospital. The inclusion criteria were: (a) a first psychotic episode meeting the Structured Clinical Interview of DSM-IV diagnostic criteria for schizophrenia; (b) age between 18 and 45 years; (c) Han Chinese ancestry; (d) total illness duration of <3 years; and (e) previous antipsychotic exposure time of <2 weeks. Exclusion criteria were: (a) DSM-IV Axis I diagnosis other than
Demographics, clinical Characteristics, and serum Anti-NMDAR Ab levels
The demographic and clinical characteristics of the participants are shown in Table 1. Age and gender were matched between patients and healthy controls (all P’s > 0.05). The patients had fewer years of education than controls (t = −2.165, P = 0.032), and years of education served as a covariate in data analyses. In the patient group, the mean age of onset of illness was 26.2 ± 7.3 years and the mean duration of illness was 13.1 ± 16.3 months. Serum anti-NMDAR Ab levels were significantly
Discussion
Our results indicated that serum anti-NMDAR Ab levels were significantly elevated in patients with schizophrenia compared with age and gender-matched controls. This is consistent with a conclusion in a previous review of seven studies comprising 1441 patients with schizophrenia and related psychoses that 115 (8%) of patients were positive for anti-NMDAR Abs (Pollak et al., 2014). These suggest that the NMDAR Abs may play a role in partially number of patients with schizophrenia (Teixeira et
Declaration of Competing Interest
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
Acknowledgments
Supports were received for this study from the National Key R & D Program of China (2016YFC1307000), Beijing Municipal Administration of Hospitals Clinical medicine Dengfeng plan funding support (DFL20151901), National Natural Science Foundation of China (81771452, 81761128021), Beijing Natural Science Foundation (7151005), and the National Institute of Health (R01MH112180 and R01MH116948).
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