Abstract
Epileptogenic Effects Of NMDAR Antibodies in a Passive Transfer Mouse Model.
Wright S, Hashemi K, Stasiak L, Bartram J, Lang B, Vincent A, Upton AL. Brain 2015:138;3159–3167.
Most patients with N-methyl D-aspartate-receptor antibody encephalitis develop seizures but the epileptogenicity of the antibodies has not been investigated in vivo. Wireless electroencephalogram transmitters were implanted into 23 C57BL/6 mice before left lateral ventricle injection of antibody-positive (test) or healthy (control) immunoglobulin G. Mice were challenged 48 h later with a subthreshold dose (40 mg/kg) of the chemo-convulsant pentylenetetrazol and events recorded over 1 h. Seizures were assessed by video observation of each animal and the electroencephalogram by an automated seizure detection programme. No spontaneous seizures were seen with the antibody injections. However, after the pro-convulsant, the test mice (n = 9) had increased numbers of observed convulsive seizures (P = 0.004), a higher total seizure score (P = 0.003), and a higher number of epileptic ‘spike’ events (P = 0.023) than the control mice (n = 6). At post-mortem, surprisingly, the total number of N-methyl D-aspartate receptors did not differ between test and control mice, but in test mice the levels of immunoglobulin G bound to the left hippocampus were higher (P50.0001) and the level of bound immunoglobulin G correlated with the seizure scores (R2 = 0.8, P = 0.04, n = 5). Our findings demonstrate the epileptogenicity of N-methyl D aspartate receptor antibodies in vivo, and suggest that binding of immunoglobulin G either reduced synaptic localization of N-methyl D-aspartate receptors, or had a direct effect on receptor function, which could be responsible for seizure susceptibility in this acute short-term model.
Commentary
Several years ago, polar bear Knut of the Berlin Zoological Garden became (with a significant help of media) a social phenomenon from the time he was a young cub being raised by zookeepers to his untimely death by drowning as a direct consequence of a seizure. Indeed, investigation of his brain determined that Knut represents another mammalian species besides humans who develop anti-N-methyl-D-aspartate (NMDA) receptor encephalitis (1).
The disorder was first reported in 2005 in women (who represent ~80% of patients) with ovarian teratomas, and it was characterized by psychiatric symptoms, memory deficits, limited consciousness, and hypoventilation. In 2007, specific autoantibodies against the NMDA receptors were discovered in patients (2) with these and additional diverse symptoms, such as seizures, catatonia, memory problems, and abnormal movements. Investigation of the anti-NMDA receptor encephalitis also provoked an interest in other synaptic autoimmune encephalitides featuring antibodies against AMPA receptors, GABA-B receptors, and leucine-rich glioma inactivated 1 protein (specific for limbic encephalitis) (3). The anti-NMDA receptor encephalitis is quite common, in prospect occupying about 4% of all encephalitides. A majority of patients present with nonspecific prodromal symptoms (headache, fever, nausea, vomiting, or upper respiratory tract symptoms), which progress into the early stage of the disorder characterized by psychiatric symptoms (anxiety, insomnia, grandiose delusions, mania, or paranoia). Hand in hand come language and memory problems. Seizures also develop during the early stage of the disease (3). While their frequency and intensity decrease with disease progression, they can resurface any time and quickly develop into status epilepticus. Later phases of the disease feature decreased responsiveness with alternating manic and catatonic periods. At this stage, autonomic symptoms are frequent, including hyperthermia, tachycardia (or bradycardia up to long-lasting cardiac pauses), hypertension (or hypotension), hypersalivation, urinary incontinence, or hypoventilation even requiring ventilation support (3). In late stages, dissociative responses to stimuli (visual vs pain) are often recorded and are reminiscent of the effects of dissociative anesthetic ketamine, which is an NMDA receptor blocker. Of all patients with anti-NMDA receptor encephalitis, about 75% recover after immunotherapy (and teratoma removal if applicable). Despite recovery, there are frequent persisting cognitive deficits (4), which are likely associated with hippocampal damage characterized as volume decreases in hippocampal subfields (5). The rest of the patients (25%) will remain severely disabled or die (3).
The current study focused on the pathogenicity of human anti-NMDA receptor antibodies. Previously, mice microinfused with human CSF positive for anti-NMDA receptor antibodies developed cognitive and behavioral problems (6) likely resulting from impairment of NMDA receptor function in the hippocampus (7). However, seizures, which frequently occur in patients with anti-NMDA receptor encephalitis, were not observed. Seizures may be also present in some patients with anti-NMDA receptor antibodies, however, without development of specific encephalitis. Thus, the authors investigated the epileptogenic properties of human anti-NMDA receptor antibodies in C57BL/6 mice.
Female mice were implanted with bilateral electrodes over the sensorimotor cortex and connected to subcutaneous wireless transmitter mediating a single channel of a bipolar EEG output. Then, two different approaches were used:
In the first experiment, the mice were stereotactically microinfused in the lateral ventricle under isoflurane anesthesia with 8 μl of purified IgG (concentration 8–40 μg/μl) from plasma of anti-NMDA receptor antibody positive patients or control subjects on day 7 post-implantation. The procedure was repeated again on day 14. EEG was then recorded to identify occurrence of seizures. The authors selected two automated and complementary techniques for EEG evaluation: 1) EEG power spectra analysis broken down into weeks (during a week after electrode implantation; during a week after the first IgG microinfusion, and during a week after the second microinfusion) and 2) EEG pattern recognition based on a library of patterns. No changes, however, were found in the EEG power spectra or the EEG pattern recognition.
It should be noted that there were several elements in the experimental design aimed to decrease the chance of finding false-positive outcomes. First, only two active electrodes were placed in the matching sites of the neocortex bilaterally. The recording signal was a differential between two active electrodes placed on the sites that have reciprocal monosynaptic connections through the corpus callosum. This leads to significant cross-correlation of the signals from both sides and, thus, a very small differential signal. This electrode arrangement also contributed to quite low overall power spectra of the EEG. Second, the EEG power spectra was calculated over quite a long period of time (1 week) that indeed decreased the chance of recording a shift in power spectra caused by a brief seizure (or a couple of those) compared with controls. Finally, EEG seizures come in different flavors, durations, and amplitudes, which may not be completely covered by pattern recognition.
In the second experiment, the authors followed seizures provoked by a subthreshold dose of 40 mg/kg intraperitoneal pentylenetetrazole injected 48 hours after the microinfusion of the anti-NMDA receptor antibody containing IgG or control IgG. This approach was selected because the first experiment did not capture spontaneous seizures. Now, the authors observed more myoclonic twitches (accompanied by epileptiform spikes) in those mice microinfused with anti-NMDA receptor antibody compared with controls. They also describe registering a larger number of brief behavioral seizure events (3–4 s duration; typical consensual duration of a seizure is >5 s), albeit without an EEG correlate. The authors then analyzed the seizure semiology in more detail, with subclassification of stage 3 seizures —into specific subtypes. In the proprietary seizure scale developed by the authors, full numeric expression would have been more convenient because it would have made possible quantitative evaluation (e.g., using nonparametric tests). Despite using an outcome-unfriendly system, the authors were able to demonstrate their major point, that is, a significant contribution of anti-NMDA receptor antibodies to increased seizure susceptibility, in other words, their proconvulsant properties.
Finally, the authors demonstrated a positive correlation between hippocampal binding of the anti-NMDA receptor antibody-positive IgG and the seizure score as well as more NMDA receptors bound with IgG from patients with anti-NMDA receptor encephalitis than with control IgG. This is an important finding because binding of antibody leads to internalization of the surface NMDA receptors and thus, to limited availability of these receptors for neurotransmission (8). However, the authors have not been able to confirm decreased expression of the surface NMDA receptors after binding of the anti-NMDA receptor antibody-positive IgG.
In conclusion, this article demonstrates the pathogenicity of human IgG containing anti-NMDA receptor antibodies in terms of increased seizure susceptibility and confirms that the antibodies are likely sufficient to cause seizures in patients with anti-NMDA receptor encephalitis. However, there are still many questions to be answered: Why does the incapacitation of NMDA receptors bring on seizures? One explanation would be a higher affinity of the anti-NMDA receptor antibody to certain brain structures. For example, if the anti-NMDA antibody binds preferentially to the striatum (and some movement disorders associated with anti-NMDA receptor encephalitis suggest that this may be the case [9]), then the proconvulsant effect can be accomplished by incapacitating the endogenous seizure-controlling network located within the basal ganglia (10). Indeed, a lack of excitation provided by excitatory neurons may lead to decreased drive onto GABAergic inhibitory neurons resulting in increased seizure susceptibility (11). Another explanation may be that the antibody preferably binds to NMDA receptors on GABAergic inhibitory neurons. In that case, is there another binding domain specific for GABA neurons required for the antibody binding? Or are the NMDA receptors on GABA neurons different from NMDA receptors on other neurons? These questions appeal for more studies at the cellular level (such as recording NMDA receptor-mediated currents on identified GABAergic and glutamatergic neurons) to recognize possible cell type-specific effects of the anti-NMDA receptor antibodies.