Teresa Moloney

Intracellular and non-neuronal targets of voltage-gated potassium channel complex antibodies

Objectives Autoantibodies against the extracellular domains of the voltage-gated potassium channel (VGKC) complex proteins, leucine-rich glioma-inactivated 1 (LGI1) and contactin-associated protein-2 (CASPR2), are found in patients with limbic encephalitis, faciobrachial dystonic seizures, Morvans syndrome and neuromyotonia. However, in routine testing, VGKC complex antibodies without LGI1 or CASPR2 reactivities (double-negative) are more common than LGI1 or CASPR2 specificities. Therefore, the target(s) and clinical associations of double-negative antibodies need to be determined. Methods Sera (n=1131) from several clinically defined cohorts were tested for IgG radioimmunoprecipitation of radioiodinated α-dendrotoxin (125I-αDTX)-labelled VGKC complexes from mammalian brain extracts. Positive samples were systematically tested for live hippocampal neuron reactivity, IgG precipitation of 125I-αDTX and 125I-αDTX-labelled Kv1 subunits, and by cell-based assays which expressed Kv1 subunits, LGI1 and CASPR2. Results VGKC complex antibodies were found in 162 of 1131 (14%) sera. 90 of these (56%) had antibodies targeting the extracellular domains of LGI1 or CASPR2. Of the remaining 72 double-negative sera, 10 (14%) immunoprecipitated 125I-αDTX itself, and 27 (38%) bound to solubilised co-expressed Kv1.1/1.2/1.6 subunits and/or Kv1.2 subunits alone, at levels proportionate to VGKC complex antibody levels (r=0.57, p=0.0017). The sera with LGI1 and CASPR2 antibodies immunoprecipitated neither preparation. None of the 27 Kv1-precipitating samples bound live hippocampal neurons or Kv1 extracellular domains, but 16 (59%) bound to permeabilised Kv1-expressing human embryonic kidney 293T cells. These intracellular Kv1 antibodies mainly associated with non-immune disease aetiologies, poor longitudinal clinical–serological correlations and a limited immunotherapy response. Conclusions Double-negative VGKC complex antibodies are often directed against cytosolic epitopes of Kv1 subunits and occasionally against non-mammalian αDTX. These antibodies should no longer be classified as neuronal-surface antibodies. They consequently lack pathogenic potential and do not in themselves support the use of immunotherapies.

IgG and complement deposition and neuronal loss in cats and humans with epilepsy and voltage-gated potassium channel complex antibodies.

Voltage-gated potassium channel complex (VGKC-complex) antibody (Ab) encephalitis is a well-recognized form of limbic encephalitis in humans, usually occurring in the absence of an underlying tumor. The patients have a subacute onset of seizures, magnetic resonance imaging findings suggestive of hippocampal inflammation, and high serum titers of Abs against proteins of the VGKC-complex, particularly leucine-rich, glioma-inactivated 1 (LGI1). Most patients are diagnosed promptly and recover substantially with immunotherapies; consequently, neuropathological data are limited. We have recently shown that feline complex partial cluster seizures with orofacial involvement (FEPSO) in cats can also be associated with Abs against VGKC-complexes/LGI1. Here we examined the brains of cats with FEPSO and compared the neuropathological findings with those in a human with VGKC-complex-Ab limbic encephalitis. Similar to humans, cats with VGKC-complex-Ab and FEPSO have hippocampal lesions with only moderate T-cell infiltrates but with marked IgG infiltration and complement C9neo deposition on hippocampal neurons, associated with neuronal loss. These findings provide further evidence that FEPSO is a feline form of VGKC-complex-Ab limbic encephalitis and provide a model for increasing understanding of the human disease.

EEG-confirmed epileptic activity in a cat with VGKC-complex/LGI1 antibody-associated limbic encephalitis

A 5-year-old, female client-owned cat presented with acute onset of focal epileptic seizures with orofacial twitching and behavioural changes. Magnetic resonance imaging showed bilateral temporal lobe hyperintensities and the EEG was consistent with ictal epileptic seizure activity. After antiepileptic and additional corticosteroid treatment, the cat recovered and by 10 months of follow-up was seizure-free without any problem. Retrospectively, antibodies to LGI1, a component of the voltage-gated potassium channel-complex, were identified. Feline focal seizures with orofacial involvement have been increasingly recognised in client-owned cats, and autoimmune limbic encephalitis was recently suggested as a possible aetiology. This is the first report of EEG, MRI and long-term follow-up of this condition in cats which is similar to human limbic encephalitis.

Persistent microglial activation and synaptic loss with behavioral abnormalities in mouse offspring exposed to CASPR2-antibodies in utero

Gestational transfer of maternal antibodies against fetal neuronal proteins may be relevant to some neurodevelopmental disorders, but until recently there were no proteins identified. We recently reported a fivefold increase in CASPR2-antibodies in mid-gestation sera from mothers of children with intellectual and motor disabilities. Here, we exposed mice in utero to purified IgG from patients with CASPR2-antibodies (CASPR2-IgGs) or from healthy controls (HC-IgGs). CASPR2-IgG but not HC-IgG bound to fetal brain parenchyma, from which CASPR2-antibodies could be eluted. CASPR2-IgG exposed neonates achieved milestones similarly to HC-IgG exposed controls but, when adult, the CASPR2-IgG exposed progeny showed marked social interaction deficits, abnormally located glutamatergic neurons in layers V–VI of the somatosensory cortex, a 16% increase in activated microglia, and a 15–52% decrease in glutamatergic synapses in layers of the prefrontal and somatosensory cortices. Thus, in utero exposure to CASPR2-antibodies led to permanent behavioral, cellular, and synaptic abnormalities. These findings support a pathogenic role for maternal antibodies in human neurodevelopmental conditions, and CASPR2 as a potential target.

Autoantibodies to glutamic acid decarboxylase in patients with epilepsy and their relationship with type 1 diabetes: a pilot study

Epilepsy is one of the most frequent neurological disorders affecting between 0.5% and 1% of the population, but in many the aetiology is unknown. A recent population-based study reported a fivefold increase in patients with type 1 diabetes mellitus (T1DM).1 Autoantibodies that recognise neuronal proteins have been identified in a number of immunotherapy-responsive seizure-related neurological disorders.2 High-titre autoantibodies to glutamic acid decarboxylase (GAD), an intracellular enzyme that catalyses the synthesis of gamma-aminobutyric acid (GABA) have been detected in neurological diseases including epilepsy although these patients show a less clear response to immunotherapies.3 ,4 GAD is also expressed by pancreatic β cells and is a major autoantigen in T1DM. Antibodies to GAD (GAD Abs) are present in up to 80% of patients with newly diagnosed T1DM, although not considered causative. There have been studies examining the incidence of GAD Abs in patients with epilepsy;5 however, we compare GAD and other Abs in patients with T1DM, with and without epilepsy. ### Study design and participants We recruited patients with T1DM (n=25) and coexistent epilepsy (T1DM/Ep) and sex-matched/age-matched patients with T1DM (n=36) from Nottingham and Sheffield Teaching Hospitals NHS Trust; the diagnoses of epilepsy and T1DM were established by specialist neurology (PM and SH) and diabetology (II) teams. Ethics approval was obtained, and written informed consent was obtained from all patients involved. ### Radioimmunoassays Voltage-gated potassium channel (VGKC)-complex antibodies were determined by immunoprecipitation of 125I-α-dendrotoxin-labelled VGKC complexes from rabbit brain extract (positive >100 pmoles/L).5 GAD65 Abs were measured using …

Glutamate receptor δ2 serum antibodies in pediatric opsoclonus myoclonus ataxia syndrome

Objective To identify neuronal surface antibodies in opsoclonus myoclonus ataxia syndrome (OMAS) using contemporary antigen discovery methodology. Methods OMAS patient serum immunoglobulin G immunohistochemistry using age-equivalent rat cerebellar tissue was followed by immunoprecipitation, gel electrophoresis, and mass spectrometry. Data are available via ProteomeXchange (identifier PXD009578). This generated a list of potential neuronal surface cerebellar autoantigens. Live cell-based assays were used to confirm membrane-surface antigens and adsorb antigen-specific immunoglobulin Gs. The serologic results were compared to the clinical data. Results Four of the 6 OMAS sera tested bound rat cerebellar sections. Two of these sera with similar immunoreactivities were used in immunoprecipitation experiments using cerebellum from postnatal rat pups (P18). Mass spectrometry identified 12 cell-surface proteins, of which glutamate receptor δ2 (GluD2), a predominately cerebellar-expressed protein, was found at a 3-fold-higher concentration than the other 11 proteins. Antibodies to GluD2 were identified in 14/16 (87%) OMAS samples, compared with 5/139 (5%) pediatric and 1/38 (2.6%) adult serum controls (p < 0.0001), and in 2/4 sera from patients with neuroblastoma without neurologic features. Adsorption of positive OMAS sera against GluD2-transfected cells substantially reduced but did not eliminate reactivity toward cerebellar sections. Conclusion Autoantibodies to GluD2 are common in patients with OMAS, bind to surface determinants, and are potentially pathogenic.

Maternal CASPR2 antibodies and neurodevelopmental disorders in the offspring: epidemiological findings and an animal model

Abstract Background CNS disorders can be caused by IgG antibodies to neuronal surface proteins, and these antibodies have the potential to cross the placenta. Since some of them target proteins involved in neurodevelopment, such as contactin-associated protein-2 (CASPR2), we hypothesised that they could alter developing neuronal circuits in utero and lead to neurodevelopmental disorders in the children. Methods A dual approach to the study was undertaken. First, we studied pregnancy serum samples from two population-based Danish cohorts: a cohort of women with postpartum psychosis and a cohort of mothers of children with mental retardation and other disorders of psychological development (MR–DPD), each with individually matched controls. We screened them for the presence of antibodies to CASPR2. Then, we assessed the effects of in-utero exposure to the antibodies in a mouse maternal-to-fetal model. Findings The combined results from these two cohorts showed that maternal antibodies to CASPR2 were associated with an increased risk of MR–DPD in the children: eight (4·4%) of 182 mothers of MR–DPD children had CASPR2 antibodies compared with only three (0·9%) of 347 mothers of children without this diagnosis (p=0·01). This association was explored by injection of mouse dams with IgG purified from CASPR2-antibody-positive patients, or from healthy controls. The mouse offspring had long-term behavioural sequelae, manifested as deficits in social interaction and social activities, and increased repetitive, non-social behaviours. When they were culled at 12 months, their brains showed abnormal migration of cortical projection neurons, increased microglial activation, and reduction in the number of glutamatergic synapses, confirming that there had been long-term changes in neurodevelopment. Interpretation CASPR2 was identified as a specific target for maternal antibodies that can alter brain development in utero, resulting in long-term behavioural deficits and permanent abnormalities at the cellular and synaptic level. These results should encourage further epidemiological and experimental studies to confirm and explore further how CASPR2 and other maternal antibodies can lead to neurodevelopmental disorders in children. Funding EC was funded by the Programme for Advanced Medical Education at the Calouste Gulbenkian Foundation. This work was also supported by the Stanley Medical Research Institute. Resources were provided by the Nuffield Department of Clinical Neurosciences.