Antibodies to Kv1 potassium channel-complex proteins leucine-rich, glioma inactivated 1 protein and contactin-associated protein-2 in limbic encephalitis, Morvan's syndrome and acquired neuromyotonia

Abstract

Antibodies that immunoprecipitate (125)I-alpha-dendrotoxin-labelled voltage-gated potassium channels extracted from mammalian brain tissue have been identified in patients with neuromyotonia, Morvan's syndrome, limbic encephalitis and a few cases of adult-onset epilepsy. These conditions often improve following immunomodulatory therapies. However, the proportions of the different syndromes, the numbers with associated tumours and the relationships with potassium channel subunit antibody specificities have been unclear. We documented the clinical phenotype and tumour associations in 96 potassium channel antibody positive patients (titres >400 pM). Five had thymomas and one had an endometrial adenocarcinoma. To define the antibody specificities, we looked for binding of serum antibodies and their effects on potassium channel currents using human embryonic kidney cells expressing the potassium channel subunits. Surprisingly, only three of the patients had antibodies directed against the potassium channel subunits. By contrast, we found antibodies to three proteins that are complexed with (125)I-alpha-dendrotoxin-labelled potassium channels in brain extracts: (i) contactin-associated protein-2 that is localized at the juxtaparanodes in myelinated axons; (ii) leucine-rich, glioma inactivated 1 protein that is most strongly expressed in the hippocampus; and (iii) Tag-1/contactin-2 that associates with contactin-associated protein-2. Antibodies to Kv1 subunits were found in three sera, to contactin-associated protein-2 in 19 sera, to leucine-rich, glioma inactivated 1 protein in 55 sera and to contactin-2 in five sera, four of which were also positive for the other antibodies. The remaining 18 sera were negative for potassium channel subunits and associated proteins by the methods employed. Of the 19 patients with contactin-associated protein-antibody-2, 10 had neuromyotonia or Morvan's syndrome, compared with only 3 of the 55 leucine-rich, glioma inactivated 1 protein-antibody positive patients (P < 0.0001), who predominantly had limbic encephalitis. The responses to immunomodulatory therapies, defined by changes in modified Rankin scores, were good except in the patients with tumours, who all had contactin-associated-2 protein antibodies. This study confirms that the majority of patients with high potassium channel antibodies have limbic encephalitis without tumours. The identification of leucine-rich, glioma inactivated 1 protein and contactin-associated protein-2 as the major targets of potassium channel antibodies, and their associations with different clinical features, begins to explain the diversity of these syndromes; furthermore, detection of contactin-associated protein-2 antibodies should help identify the risk of an underlying tumour and a poor prognosis in future patients.

Figure 1

VGKC-antibodies immunoprecipitate VGKCs from brain extracts but do not bind directly to Kv1 subunits. (A) VGKC-antibody levels (pM), measured by immunoprecipitation of ¹²⁵I-α-DTX-VGKCs from rabbit brain extracts, in 96 patients, divided according to the clinical diagnoses (Table 1), and from 70 healthy or other disease controls. The cut-off (100 pM) represents the mean plus three standard deviations of the healthy control values. (B) Binding of antibodies to the surface of HEK293 cells transfected with cDNA for the Kv1.1 subunit; the cells were co-transfected with EGFP to identify those cells taking up the cDNA. Positive staining was found with a rabbit antibody raised against the extracellular domain of Kv1.1 (anti-Kv1.1), but VGKC-antibody positive sera did not show any staining (lower panels). Magnification × 1000 (C) Voltage-dependent currents measured in Kv1.1 transfected HEK293 cells were substantially reduced by the neurotoxin, α-DTX (1 µM). (D) Sera (1:100 diluted) from six healthy individuals (total 17 cells recorded) or from three individual patients with VGKC-antibodies (n = number of cells examined for each patient) had no effect on voltage-dependent currents in Kv1.1 transfected HEK293 cells (Table 2). Ab = antibody; HC = healthy control.

Figure 2

VGKCs are not the targets for the VGKC-antibodies. (A) A digitonin-extract was prepared of HEK293 cells that had been cotransfected with cDNAs for Kv1.1, 1.2, 1.6 and β2. The extract was labelled with ¹²⁵I-α-DTX. Rabbit antibodies to each of the Kv1 subunits, but only three of the 96 VGKC-antibody positive sera, immunoprecipitated the ¹²⁵I-α-DTX-labelled Kv1 complexes, showing that 93/96 of the patients’ antibodies did not bind the Kv1s at detectable levels. (B) The effect of a harsher detergent on VGKC-complexes. Addition of increasing amounts of sodium dodecyl sulphate showed that immunoprecipitation by the patients’ antibodies was reduced at very low concentrations of sodium dodecyl sulphate, in comparison with immunoprecipitation by the antibodies to Kv1.1 or 1.2, suggesting that the patients’ binding sites were easily dissociated from the ¹²⁵I-α-DTX-labelled Kv1s. (C) Immunoprecipitation of ¹²⁵I-α-DTX-labelled VGKCs extracted from brain tissue with antibodies directed against individual Kv1 subunits, Lgi1, Caspr2 and contactin-2. Each of the antibodies immunoprecipitated significant but variable quantities of ¹²⁵I-α-DTX labelled rabbit brain Kv1s indicating that Lgi1, Caspr2 and contactin-2 were complexed with the Kv1s. SDS = sodium dodecyl sulphate.

Figure 3

Caspr2-antibodies in VGKC-antibody positive patients. (A) HEK293 cells were transfected with Caspr2-EGFP. A VGKC-antibody positive serum (1:100) bound to the surface of the Caspr2-EGFP-expressing cells (green), whereas healthy serum (HC) did not bind. Strong binding (red) was found in 19 of the 96 VGKC-antibody positive sera. (B) Immunoprecipitation of Caspr2-EGFP from digitonin extracts of the transfected cells by 5 µl of serum was also positive in 19 of the 96 sera, compared with sera from other neurological disease controls (OND; multiple sclerosis, myasthenia with thymomas, non-VGKC-antibody associated encephalopathies) or healthy individuals (controls). (C) There was a highly positive correlation between the binding to Caspr2-EGFP and binding to ¹²⁵I-α-DTX-labelled Kv1s. For these data, the Caspr2-EGFP immunoprecipitation was performed with 1 µl of each serum to obtain more quantitative results. (D) Caspr2-antibody positive serum IgGs (detected with fluorescein-anti-human IgG, green) also bound strongly to the surface of live cultured hippocampal neurons, 18 days in culture; the neurons were subsequently fixed, permeabilized and stained for MAP2 (red). The bar represents 25 µm.

Figure 4

Lgi1-antibodies are present in many VGKC-antibody positive patients. (A) HEK293 cells were co-transfected with cDNAs for Lgi1 and EGFP (green) and incubated with patient sera (1:20). A representative image of binding of a limbic encephalitis VGKC-antibody positive serum to the cells, detected with anti-human IgG (red). Note that the red stain is found not only on the Lgi1/EGFP transfected cells, but also on the surface of non-transfected cells. Similar results were found in 46 of the 96 patient sera, but were not observed with healthy control sera (HC). (B) Immunoadsorption of the VGKC-antibody positive sera against Lgi1-transfected cells, but not against Kv1-transfected cells, substantially reduced immunoprecipitation of ¹²⁵I-α-DTX–VGKC complexes from rabbit brain extracts. (C) Lgi1-positive sera binding to a live hippocampal neuron detected with fluorescein-anti-human IgG (green); the neurons were subsequently fixed, permeabilized and stained for MAP2 (red). The bar represents 25 µm. (D) To confirm that Lgi1 was the target for the antibody binding shown in A, cDNA for Lgi1 was fused to cDNA for the transmembrane domain and cytoplasmic tail of Caspr2. This construct expressed well in HEK293 cells and provided a direct assay for Lgi1 antibodies. Nine additional sera (one illustrated here) bound to these cells; but there was no binding of healthy control sera or Caspr2-antibody positive sera.

Figure 5

Expression pattern of Caspr2 and Lgi1 in the CNS. Black and white images of fixed hippocampal (A and B) and cerebellar (C and D) sections double stained with specific antibodies to Caspr2 (red), Lgi1 (green) and Kv1.1 (green) as indicated in the merged images in the right column where cell nuclei are stained with DAPI (blue). In the CA3 area of the hippocampus, Caspr2 (A and B) is strongly expressed in the stratum radiatum (rad), whereas Kv1.1 (A) and Lgi1 (B) are most prominent in the mossy fibre layer (mf) where Caspr2 is almost absent. In the cerebellum, Caspr2 (C and D) is expressed in the molecular (mol) and granule cell layers (GCL), but not in the pinceau (green arrows) where Kv1.1 (C) as well as Lgi1 (D) are strongly expressed. Scale bar = 20 μm. pyr = pyramidal cell layer; PC = Purkinje cell layer.

Figure 6

Binding patterns of Caspr2 and Lgi1 immunoreactive sera in the CNS. Images of fixed hippocampal (A and B) and cerebellar (C and D) sections, or sciatic nerve teased fibres (E–G), immunostained with sera (green) from representative patients with limbic encephalitis/Morvan’s syndrome or healthy controls (far right column in A–D and G) as indicated, combined with specific antibodies (red) to Caspr2 (A and C), Lgi1 (B and D) or Caspr (which localizes to the paranode, E–G). For the limbic encephalitis/Morvan’s syndrome, sera in A–D channels are shown separately as black and white images, as well as merged images to demonstrate reactivity of the sera, while for the control sera only merged images are shown. Cell nuclei are stained with DAPI (blue). In the CA3 area of the hippocampus Serum 6 (A) binds strongly to the stratum radiatum (rad) where Caspr2 is prominently expressed (A) but not to the mossy fibre layer (mf) where Caspr2 is almost absent. In contrast, Serum 1 (B) binds to the mf more than the rad, colocalizing with Lgi1. In the cerebellum, Serum 6 (C) colocalizes with Caspr2 in the molecular (mol) and granule cell layers (GCL), but does not bind to the pinceau where Lgi1 is strongly expressed (red arrows in D) but Caspr2 is absent. In contrast to Serum 6, Serum 1 shows strong binding to the pinceau (green arrows in D) colocalizing with Lgi1 (insets in D show the pinceau at higher magnification). Control sera show no specific binding in CA3 or cerebellum (A–D). In E–G staining of sciatic nerve teased fibres with antibodies to the paranodal marker Caspr (distinct from Caspr2) and patient serum, shows that Caspr2-reactive Serum 6 strongly labels the juxtaparanodes that are known to express Caspr2 but not significantly Lgi1 (green arrows in E), whereas Lgi1-reactive Serum 1 (F), as well as a control serum (G), shows no specific binding. Scale bars: A–D = 20 μm, E–G = 10 μm. Serum 1 was from a typical Lgi1-antibody positive limbic encephalitis patient and Serum 6 was from a Caspr2-antibody positive Morvan’s syndrome patient. Pyr = pyramidal cell layer; PC = Purkinje cell layer.

Figure 7

Clinical scores before and after treatments in patients with Caspr2- or Lgi1-antibodies. (A) Modified Rankin scores pre- and post-immunotherapies in patients with Caspr2 antibodies with or without tumours. Many of the patients without tumours improved but those with tumours often deteriorated, and four died (modified Rankin scores = 6). (B) Modified Rankin scores in patients with Lgi1-antibodies before and after immunotherapies. The modified Rankin scores were significantly reduced following treatments. Only one Lgi1-antibody positive patient died (modified Rankin scores = 6), and their death was unrelated to the clinical syndrome. The Modified Rankin Scores (0 = asymptomatic patient; 1 = symptoms do not interfere with lifestyle; 2 = symptoms lead to some restriction of lifestyle but do not prevent totally independent existence; 3 = symptoms significantly interfere with lifestyle or prevent totally independent existence; 4 = symptoms clearly prevent independent existence, although patient does not need constant attention day and night; 5 = severe disability, with patient totally dependent and requiring constant attention day and night; 6 = death due to the condition) ranges from 0 (normal) to 6 (death) (Graus et al., 2001).