Antibodies to the GABA(B) receptor in limbic encephalitis with seizures: case series and characterisation of the antigen

Abstract

Background: Some encephalitides or seizure disorders once thought idiopathic now seem to be immune mediated. We aimed to describe the clinical features of one such disorder and to identify the autoantigen involved.

Methods: 15 patients who were suspected to have paraneoplastic or immune-mediated limbic encephalitis were clinically assessed. Confocal microscopy, immunoprecipitation, and mass spectrometry were used to characterise the autoantigen. An assay of HEK293 cells transfected with rodent GABA(B1) or GABA(B2) receptor subunits was used as a serological test. 91 patients with encephalitis suspected to be paraneoplastic or immune mediated and 13 individuals with syndromes associated with antibodies to glutamic acid decarboxylase 65 were used as controls.

Findings: All patients presented with early or prominent seizures; other symptoms, MRI, and electroencephalography findings were consistent with predominant limbic dysfunction. All patients had antibodies (mainly IgG1) against a neuronal cell-surface antigen; in three patients antibodies were detected only in CSF. Immunoprecipitation and mass spectrometry showed that the antibodies recognise the B1 subunit of the GABA(B) receptor, an inhibitory receptor that has been associated with seizures and memory dysfunction when disrupted. Confocal microscopy showed colocalisation of the antibody with GABA(B) receptors. Seven of 15 patients had tumours, five of which were small-cell lung cancer, and seven patients had non-neuronal autoantibodies. Although nine of ten patients who received immunotherapy and cancer treatment (when a tumour was found) showed neurological improvement, none of the four patients who were not similarly treated improved (p=0.005). Low levels of GABA(B1) receptor antibodies were identified in two of 104 controls (p<0.0001).

Interpretation: GABA(B) receptor autoimmune encephalitis is a potentially treatable disorder characterised by seizures and, in some patients, associated with small-cell lung cancer and with other autoantibodies.

Funding: National Institutes of Health.

Figure 1. MRI of a patient with GABA_B receptor antibodies and limbic encephalitis

Axial fluid-attenuated inversion recovery (FLAIR) MRI from patient 1 at presentation (A) showed increased signal in the medial temporal lobes, which was more pronounced on the left. Repeat study at 1 month (B) showed improvement of the FLAIR signal that remained stable at 3 months and 9 months (C, D), with development of mild generalised atrophy (the patient received standard whole-brain radiation therapy as prophylaxis for small-cell lung cancer metastases).

Figure 2. Immunolabelling of rat brain with patients' antibodies

Sagittal section of rat brain immunolabelled with CSF of a patient with limbic encephalitis (A) and a control individual (E). Note the extensive staining in A of the neuropil of thalamus (B), hippocampus (C), cerebellum (D), and cerebral cortex. DG=dentate gyrus. M=molecular layer. P=Purkinje cell layer. G=granular cell layer. Avidin–biotin–peroxidase method; sections counterstained with haematoxylin.

Figure 3. Culture of rat hippocampal neurons incubated (live, non-permeabilised) with the CSF of a patient with limbic encephalitis and a control individual

Note the intense punctate reactivity of patient's antibodies with cell surface antigens (A) and the absence of reactivity in the control (B); nuclei of neurons stained with 4',6-diamidino-2-phenylindole (DAPI). The surface antigens were precipitated using the antibodies within the patient's serum, and then electrophoretically separated and visualised with EZBlue (C). Patient's antibodies (P) precipitated two main protein bands at about 105 kDa and 90 kDa; these bands are not seen in the precipitate using serum from a control individual (N). Sequencing of the 105 kDa band by use of mass spectrometry showed it contained the B1 and B2 subunits of the GABA_B receptor (webappendix). The 90 kDa and other smaller bands were proteolytic fragments and patient's IgG products. Subsequent transfer of the gel to nitrocellulose and immunoblotting with antibodies specific for each of the GABA_B (D) subunits confirmed that patient's antibodies precipitated the B1 and B2 subunits (105 kDa) and that the 90 kDa band was a proteolytic fragment of B1.

Figure 4. Confocal image of a cultured triple labelled embryonic rat hippocampal neuron

Patient's antibodies are in green, a guineapig polyclonal antibody against an intracellular epitope of the GABA_B1 receptor is in red, and an antibody to the presynaptic marker Bassoon is in blue (A). Area of dendrite from the same neuron showing patient's antibody staining (B), guineapig polyclonal GABA_B1 receptor antibody staining (C), both patient and guineapig antibody staining (D), and triple staining (E). The colocalisation of labelling of the dendrites of 23 neurons was quantified (F). This suggests that patients' antibodies bind both synaptic and extrasynaptic GABA_B receptors.

Figure 5. Detection of antibodies to the GABA_B1 subunit using a HEK293 cell-based assay

HEK293 cells transfected with the GABA_B1 receptor subunit show reactivity with CSF from a patient with limbic encephalitis (A) and a polyclonal antibody against the B1 subunit of the GABA_B receptor (B); both reactivities are merged in C. Similarly transfected cells do not react with CSF from a control individual (D) but do show reactivity with a polyclonal antibody against the B1 subunit of the GABA_B receptor (E); reactivities merged in F. Immunofluorescent method.