LGI1 antibodies alter Kv1.1 and AMPA receptors changing synaptic excitability, plasticity and memory

Abstract

Leucine-rich glioma-inactivated 1 (LGI1) is a secreted neuronal protein that forms a trans-synaptic complex that includes the presynaptic disintegrin and metalloproteinase domain-containing protein 23 (ADAM23), which interacts with voltage-gated potassium channels Kv1.1, and the postsynaptic ADAM22, which interacts with AMPA receptors. Human autoantibodies against LGI1 associate with a form of autoimmune limbic encephalitis characterized by severe but treatable memory impairment and frequent faciobrachial dystonic seizures. Although there is evidence that this disease is immune-mediated, the underlying LGI1 antibody-mediated mechanisms are unknown. Here, we used patient-derived immunoglobulin G (IgG) antibodies to determine the main epitope regions of LGI1 and whether the antibodies disrupt the interaction of LGI1 with ADAM23 and ADAM22. In addition, we assessed the effects of patient-derived antibodies on Kv1.1, AMPA receptors, and memory in a mouse model based on cerebroventricular transfer of patient-derived IgG. We found that IgG from all patients (n = 25), but not from healthy participants (n = 20), prevented the binding of LGI1 to ADAM23 and ADAM22. Using full-length LGI1, LGI3, and LGI1 constructs containing the LRR1 domain (EPTP1-deleted) or EPTP1 domain (LRR3-EPTP1), IgG from all patients reacted with epitope regions contained in the LRR1 and EPTP1 domains. Confocal analysis of hippocampal slices of mice infused with pooled IgG from eight patients, but not pooled IgG from controls, showed a decrease of total and synaptic levels of Kv1.1 and AMPA receptors. The effects on Kv1.1 preceded those involving the AMPA receptors. In acute slice preparations of hippocampus, patch-clamp analysis from dentate gyrus granule cells and CA1 pyramidal neurons showed neuronal hyperexcitability with increased glutamatergic transmission, higher presynaptic release probability, and reduced synaptic failure rate upon minimal stimulation, all likely caused by the decreased expression of Kv1.1. Analysis of synaptic plasticity by recording field potentials in the CA1 region of the hippocampus showed a severe impairment of long-term potentiation. This defect in synaptic plasticity was independent from Kv1 blockade and was possibly mediated by ineffective recruitment of postsynaptic AMPA receptors. In parallel with these findings, mice infused with patient-derived IgG showed severe memory deficits in the novel object recognition test that progressively improved after stopping the infusion of patient-derived IgG. Different from genetic models of LGI1 deficiency, we did not observe aberrant dendritic sprouting or defective synaptic pruning as potential cause of the symptoms. Overall, these findings demonstrate that patient-derived IgG disrupt presynaptic and postsynaptic LGI1 signalling, causing neuronal hyperexcitability, decreased plasticity, and reversible memory deficits.

Figure 1

Patient's LGI1 IgG blocks the binding of soluble LGI1 to ADAM23 and ADAM22. (A) Top: Coomassie staining of proteins contained in the medium of HEK293 LGI1-expressing cells (lanes 2 and 3) and HEK293 cells not expressing LGI1 (lanes 4 and 5) (total protein load in lanes 2 and 4, 25 μg; and in lanes 3 and 5, 12.5 μg). Bottom: Immunoblot of protein preparations shown in the upper Coomassie panel using a polyclonal LGI1 antibody; note that LGI1 is only present in the medium derived from HEK LGI1-expressing cells. (B) HEK293 cells expressing ADAM23 or ADAM22 (in magenta) bind soluble LGI1 (in green) present in the medium of HEK293 cells expressing LGI1 (B, top row) but not present in control medium (B, second row). When the medium with soluble LGI1 is preincubated with a representative patient's LGI1 IgG the binding of LGI1 to ADAM23 or ADAM22 is abrogated (B, third row); in contrast, no blocking of the binding is observed when the medium is preincubated with control IgG (B, fourth row). Scale bar = 20 μm.

Figure 2

Reactivity of a patient's serum with deletion and chimeric constructs of LGI1. Serum from a representative patient with anti-LGI1 encephalitis shows predominant antibody reactivity (in red) with the full-length sequence of LGI1 (Clone 1), and the constructs that contain the LRR1 (Clone 3) and EPTP1 (Clone 4) domains. In contrast, the LRR3 and EPTP3 domains contained in the full-length LGI3 (Clone 2) were not recognized by patient's antibodies. Scale bar = 20 μm.

Figure 3

Patient-derived LG1 IgG causes a decrease of total cell surface and synaptic K_v1.1 clusters in the hippocampus. (A) Sagittal section of hippocampus of a representative mouse infused with patient-derived LGI1 IgG demonstrating the immunostaining of K_v1.1, Bassoon, and the merging reactivities. Squares in ‘Analysis’ indicate the analysed subregions in CA1, CA3, and dentate gyrus for each animal. Scale bar = 200 μm. (B) 3D projection and analysis of the density of total clusters of K_v1.1 and Bassoon, and synaptic clusters of K_v1.1 (defined as K_v1.1 clusters that co-localized with Bassoon) in one of the indicated CA3 subregions (A, ‘Analysis’). Merged images (B, top and bottom left; K_v1.1 green, and Bassoon red) were post-processed and used to calculate the density of the indicated clusters (density = spots/μm³). Scale bar = 2 μm. (C–E) Quantification of the density of total (C) and synaptic (D) K_v1.1 clusters, and Bassoon clusters (E) in a pooled analysis of hippocampal subregions (CA1, CA3, dentate gyrus) in animals treated with patient-derived LGI1 IgG (red) or control IgG (black) on the indicated days. Mean density of clusters in control IgG treated animals was defined as 100%. Data are presented as mean ± SEM. For each time point five animals infused with patient-derived LGI1 IgG and five with control IgG were examined. Significance of treatment effect was assessed by two-way ANOVA with an alpha-error of 0.05 (asterisks) and post hoc testing with Sidak-Holm adjustment. **P < 0.01, ***P < 0.001, ****P < 0.0001.

Figure 4

Patient-derived LG1 IgG causes a decrease of total cell surface and synaptic AMPAR clusters in the hippocampus. (A) 3D projection and analysis of the density of total clusters of GluA1 AMPAR and PSD95, and synaptic clusters of AMPAR (defined as AMPAR clusters that co-localized with PSD95) in one of the CA3 subregions indicated in Fig. 3A, ‘Analysis’. Merged images (A, top and bottom left; GluA1 green, and PSD95 red) were post-processed and used to calculate the density of the indicated clusters (density = spots/μm³). Scale bar = 2 μm. (B–D) Quantification of the density of total (B) and synaptic (C) GluA1 AMPAR clusters, and PSD95 clusters (D) in a pooled analysis of hippocampal subregions (CA1, CA3, dentate gyrus) in animals treated with patient-derived LGI1 IgG (red) or control IgG (black) on the indicated days. Mean density of clusters in control IgG treated animals was defined as 100%. Data are presented as mean ± SEM. For each time point five animals infused with patient-derived IgG and five with control IgG were examined. Significance of treatment effect was assessed by two-way ANOVA with an alpha-error of 0.05 (asterisks) and post hoc testing with Sidak-Holm adjustment. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Figure 5

Patient-derived LGI1 IgG increases presynaptic release probability. (A) Example whole-cell recordings of unitary EPSCs of CA1 pyramidal neurons after minimal stimulation of Schaffer collateral axons. Five traces of the same cell are shown for each experimental group. (B) Failure rate of unitary events is decreased in neurons of LGI1 IgG treated mice similar to neurons of control IgG treated mice after application of DTX. Failure rate is unchanged in the LGI1 IgG group upon application of DTX (n_{control IgG} = 8; n_{control IgG+DTX} = 7; n_{LGI1 IgG} = 10; n_{LGI1 IgG+DTX} = 8; box plots show the median, 25th, and 75th percentiles; whiskers indicate the 10th and 90th percentiles, outlying points are depicted as dots; one-way ANOVA and Bonferroni post hoc test. *P < 0.05). (C–E) Peak amplitude of unitary minimally stimulated EPSCs (D) and paired pulse facilitation with an interstimulus interval of 50 ms (E) is similar in CA1 neurons of all experimental groups (synaptic failures are excluded). Exemplary average traces of individual cells are shown in C.

Figure 6

Patients' LGI1 IgG increases excitatory synaptic transmission. (A and B) Evoked excitatory postsynaptic currents (eEPSCs) in dentate gyrus granule cells upon incremental stimulation of medial perforant path (MPP) afferent fibres are increased in acute brain slices of mice that received patient-derived LGI1 IgG. Example traces of an individual recording are shown in A. In/out characteristics of granule cell neurons of control IgG and patient-derived LGI1 IgG treated mice (B; n_{control IgG} = 13; n_{LGI1 IgG} = 15; data are presented as mean ± SEM; two-way ANOVA and Holm-Sidak post hoc test. ***P < 0.001). (C and D) Peak amplitude of eEPSCs (supramaximal stimulation; example traces in C) is increased after infusion of antibodies against LGI1, whereas rise and decay time is unchanged (n_{control IgG} = 13; n_{LGI1 IgG} = 15; box plots show the median, 25th, and 75th percentiles; whiskers indicate the 10th and 90th percentiles, outlying points are depicted as dots; unpaired t-test. **P < 0.01). (E and F) Paired pulse facilitation with an interstimulus interval of 50 ms is reduced in granule cell neurons from mice after infusion of patient-derived LGI1 IgG indicating increased release probability. Example traces in E; dashed lines indicate peak amplitude of the first pulse (n_{control IgG} = 11; n_{LGI1 IgG} = 13; box plots show the median, 25th, and 75th percentiles; whiskers indicate the 10th and 90th percentiles, outlying points are depicted as dots; unpaired t-test. *P < 0.05).

Figure 7

Patient-derived LGI1 IgG alters synaptic plasticity. (A) Example traces of individual fEPSP recordings in the CA1 region before (black traces) and after (red traces) theta burst stimulation (TBS) of Schaffer collateral afferents. Potentiation of fEPSPs is reduced in brain slices of mice that received patient-derived LGI1 IgG without further changes upon application of DTX. (B) Time course of LTP after theta-burst stimulation (arrow), demonstrates persistent reduction of fEPSP slope values in slices of mice after infusion of patient-derived LGI1 IgG indicating disturbed synaptic plasticity. (C) Quantification of fEPSP slope change is decreased in LGI1 IgG infused mice. Application of DTX does not alter fEPSP slope values in LGI1 and control IgG infused mice, respectively (n_{control IgG} = 14; n_{control IgG+DTX} = 9; n_{LGI1 IgG} = 18; n_{LGI1 IgG+DTX} = 8; box plots show the median, 25th, and 75th percentiles; whiskers indicate the 10th and 90th percentiles, outlying points are depicted as dots; one-way ANOVA and Bonferroni post hoc test. *P < 0.05, **P < 0.01).

Figure 8

Cerebroventricular infusion of patient-derived LGI1 IgG causes decrease of memory. Novel object recognition index in mice treated with patient-derived LGI1 IgG (red, n = 16 mice) or control IgG (grey, n = 14 mice). A high index indicates better memory. Data are presented as mean ± SEM. *P < 0.05; **P < 0.01.