Original article
Antibodies to AMPA receptors in Rasmussen's encephalitis

https://doi.org/10.1016/j.ejpn.2015.12.011Get rights and content

Highlights

  • Fifty two patients with Rasmussen's Encephalitis were tested for antibodies to AMPA receptors using cell-based assays.

  • Two patients (3.8%) had antibodies to AMPA receptors comprising of GluA2/3 co-expressed together.

  • No sera showed reactivity to the singly transfected receptors (GluA1, 2 or 3) or to co-expressed GluA1/2.

  • Ten patients (including both patients with antibodies that bound GluA2/3) bound to the surface of cultured neurones.

  • The antibodies detected to GluA2/3 may represent a biomarker of the inflammatory nature of the disease.

Abstract

Rasmussen's encephalitis is a rare progressive childhood disorder characterized by frequent severe seizures, hemiparesis, encephalitis and mental deterioration, and associated with severe unilateral neuroinflammation. Autoantibodies, particularly to the GluA3 subtype of the alpha-amino-3-hydroxy-5-methyl-isoxazole-4-propinonic acid receptor (AMPAR) were reported in the 1990s but not always confirmed. To explore further, sera from 52 patients with Rasmussen's encephalitis were tested by cell-based assays for antibodies to AMPAR GluA1/2/3, N-methyl-d-aspartate (NMDA NR1/2b), γ-aminobutyric acid type A and B (GABAAR α1/γ2/β2 and GABABR b1/b2) receptors, for potassium channel complex proteins, and for binding to live cortical and hippocampal neuronal cultures. Two patients' sera (3.8%) bound to HEK cells co-transfected with the GluA2 and GluA3 subunits. One additional patient had a low level of VGKC-complex antibodies. These three, and seven additional, sera bound to hippocampal cultures. No other antibodies were detected. Thus, despite the rarity of GluA2/3 antibodies, 10 patients (19.2%) had evidence of antibodies to neuronal antigens. Whether these antibodies play a primary role in RE, or appear secondary to the neuro-inflammatory damage in this highly destructive disease, requires further study.

Introduction

Rasmussen's encephalitis (RE) is a severe epileptic syndrome, characterized by unihemispheric brain dysfunction and tissue loss.1 An inflammatory histopathology, neurological impairment and intractable focal seizures, including epilepsia partialis continua (EPC), are common clinical features. Magnetic Resonance Imaging (MRI) of patients with RE demonstrates the spread of cortical inflammation throughout the hemisphere. The disease almost always leads to hemiparesis and atrophy (reviewed in2). Neuronal cell loss, microglial nodules and T cell infiltrates are common pathological features of the affected cerebral cortex.2, 3 Patients with RE experience frequent seizures, and these are poorly responsive to anti-epileptic drugs (AEDs). Some patients show a positive response to immunotherapies such as plasma exchange, and tacrolimus.2, 4

In 1994, rabbits immunized with a recombinant fusion protein of GluA3 (then termed GluR3, an alpha-amino-3-hydroxy-5-methyl-isoxazole-4-propinonic acid receptor (AMPAR) subunit) developed GluA3 antibodies and seizures, and their cerebral histopathology showed features that resembled RE. Five children with RE were found to have similar antibodies, and one child showed a very good response to plasma exchange, suggesting that GluA3 antibodies (Abs) might play a pathogenic role.4 However, GluA3 Abs, detected by enzyme linked immunosorbent assays (ELISAs) using short peptide sequences of GluA3, were subsequently reported not only in RE patients but also in other epilepsies and controls, and further studies using a range of methods did not identify GluA3 Abs at that time.5

In the last 15 years a variety of Abs to neuronal proteins have been identified in central nervous system (CNS) disorders using cell-based and other assays designed to detect Abs that bind to conformational cell-surface exposed antigens,6 including the AMPA receptor7 AMPA receptors are of particularly interest as they mediate excitatory transmission in the central nervous system and have been shown to be critical to the generation and spreading of epileptic activity.8 Here we used these approaches to examine/re-examine 52 RE patients for the newly described Abs using these techniques and investigated further the role of antibodies to the AMPA receptor.

Section snippets

Patients details

Informed consent was obtained from all patients, or their parents, seen in Oxford (see5), Bonn or other neurological centers over many years with approval from the local ethics committee as appropriate at the time. A diagnosis of Rasmussen's encephalitis was made using standard criteria.9. Samples from children (<18 years) with other forms of epilepsy (n = 106) and healthy controls (n = 112) were also tested, as approved by the Oxfordshire Regional Ethical Committee A (07/Q1604/28).

Diagnostic assays

In this

Patient cohort

Fifty two patients with RE (n = 38 childhood-onset and n = 14 adult-onset) were studied (see Table 1). The median age of diagnosis of the childhood-onset patients was 7 years (range: 1–18), and of adult patients was 32 years (range: 19–41). The median duration of disease at time of sampling was 2.2 years (range: 0.1–27). Two patients had paired serum and CSF samples and one additional patient had an unpaired CSF. Results were compared to samples from disease and healthy controls.

Antibody results

Although some

Discussion

In the 1990s a number of reports implicated Abs to AMPA receptors, particularly to GluA3, in children with RE, but the techniques used were not optimal and not all studies confirmed their disease specificity or presence.4, 5 However, using live cell based assays, by which only antibodies to cell-surface epitopes are detected, we identified Abs to AMPAR in two RE patients, both with typical RE courses. The Abs bound only when GluA2 and GluA3 were co-expressed together (GluA2/3), suggesting that

Conclusions

Antibodies to GluA2/3 have only been found in the sera of limited number of patients and only when the two subunits (GluA2 and GluA3) of the AMPA receptor were co-expressed together. No antibodies were detected that recognized the singly transfected cells or the GluA1 and GluA2 co-transfection. However, ten sera (19%) bound to the surface of live neurons. It is unclear whether these antibodies play a primary role in the pathogenesis of RE but may appear secondary to inflammatory events that

Authors contributions

Manuscript drafting and critical revision of manuscript: AN, AV, CEE, PW, CGB, BL.

Identification of patients, clinical information: CGB, CEE.

Data Acquisition: AN, PP, LC, PW, BL.

Funding

BL receives funding (P1201) from Epilepsy Research UK (ERUK) and Pablove Foundation, the Oxford NIHR Biomedical Research Centre and the National Health Service National Specialised Commissioning Group for Neuromyelitis Optica (PW, AV).

Conflict of interest

AV and the Nuffield Department of Clinical Neurology Neurosciences in Oxford receive royalties and payments for antibody assays and AV is the named inventor on patent application WO/2010/046716 entitled 'Neurological Autoimmune Disorders'. The patent has been licensed to Euroimmun AG for the development of assays for LGI1 and other VGKC-complex antibodies. PW and BL are co-inventors and have received royalties. PW has received speaker honoraria from Biogen Idec and Euroimmun AG. CEE received

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