Human cerebrospinal fluid monoclonal CASPR2 autoantibodies induce changes in electrophysiology, functional MRI, and behavior in rodent models

Abstract

Anti-contactin associated protein receptor 2 (CASPR2) encephalitis is a severe autoimmune encephalitis with a variable clinical phenotype including behavioral abnormalities, cognitive decline, epileptic seizures, peripheral nerve hyperexcitability and neuropathic pain. The detailed mechanisms of how CASPR2 autoantibodies lead to synaptic dysfunction and clinical symptoms are largely unknown. Aiming for analyses from the molecular to the clinical level, we isolated antibody-secreting cells from the cerebrospinal fluid of two patients with CASPR2 encephalitis. From these we cloned four anti-CASPR2 human monoclonal autoantibodies (mAbs) with strong binding to brain and peripheral nerves. All were highly hypermutated and mainly of the IgG4 subclass. Mutagenesis studies determined selective binding to the discoidin domain of CASPR2. Surface plasmon resonance revealed affinities with dissociation constants K_D in the pico- to nanomolar range. CASPR2 mAbs interrupted the interaction of CASPR2 with its binding partner contactin 2 in vitro and were internalized after binding to CASPR2-expressing cells. Electrophysiological recordings of rat hippocampal slices after stereotactic injection of CASPR2 mAbs showed characteristic afterpotentials following electrical stimulation. In vivo experiments with intracerebroventricular administration of human CASPR2 mAbs into mice and rats showed EEG-recorded brain hyperexcitability but no spontaneous recurrent seizures. Behavioral assessment of infused mice showed a subtle clinical phenotype, mainly affecting sociability. Mouse brain MRI exhibited markedly reduced resting-state functional connectivity without short-term structural changes. Together, the experimental data support the direct pathogenicity of CASPR2 autoantibodies. The minimally invasive EEG and MRI techniques applied here may serve as novel objective, quantifiable tools for improved animal models, in particular for subtle neuropsychiatric phenotypes or repeated measurements.

Keywords: Autoantibodies; Autoimmune encephalitis; CASPR2; Human monoclonal antibody; Mouse MRI.

Fig. 1

CASPR2 reactivity of isolated monoclonal antibodies. Cell based assay CASPR2 reactivity for HK 187–194 (A), HL 187–188 (B), HL 187–180 (C) and HK 219–142 (D), with human IgG shown in green, and c-myc-DDK tagged CASPR2 in red. Validation of CASRP2 reactivity shown in intrathecally infused mouse hippocampus (E) mouse teased sciatic nerves at 40x magnification for HK 187–194, with human IgG shown in green, and K_v1.2 shown in red (F).

Fig. 2

Binding characterization of CASPR2 reactive monoclonal antibodies. Schematic of CASPR2 protein, indicating the location of each domain and subsequent generated deletion constructs, shown below is reactivity to each corresponding construct through cell based assay, with green showing human IgG (A). Schematic of the discoidin isolation construct with the reactivity of each mAb in green shown below (B). Binding strength of the monoclonal antibodies as determined through a solid phase binding assay using human CASPR2 protein. Antibody concentration used ranged from 50 to 0.05 ug/ml. Bars indicate mean ± SEM (C). Affinity of three antibodies as determined by surface plasma resonance, using human CASPR2 protein, shown with kD values (D). Inhibition of contactin-2 binding to CASPR2 determined in solid phase binding assays (n = 3), here shown in percentage of contactin-2 inhibited compared to condition without antibody. Each antibody is shown in a range of concentrations, from 50 to 0.05 ug/ml. Bars indicate mean ± SEM (E). Internalization of pHrodo-coupled anti-CASPR2 mAbs after 4 h and 24 h incubation on CASPR2-transfected HEK293T cells. Untransfected cells were used as controls. Scale bar indicates 10 μm (F).

Fig. 3

Electrophysiological effects of CASPR2 reactive monoclonal antibodies. fEPSP amplitude of ex vivo hippocampal slices measured after a range of stimuli intensities (25-200μA), for CASPR2 mab treated and control treated tissue (A). The relative fEPSP amplitude shown during, basal synaptic transmission input-out relationship (I/O_pre) and potentiated synaptic transmission input-out relationship (I/O_post) with STP induction at minute 10 (B). Representative fEPSP traces from a control and a CASPR2-mAb-injected animal, with epileptiform afterpotentials indicated with asterisks (C). Number of afterpotentials measured during I/O_pre, STP induction and I/O_post, with isignificant differences in the STP and I/O_post conditions between CASPR2 and control treated tissue (P<0,01 Mann-Whitney test) (D). The paired-pulse ratio (PPR) showed a significant decrease after STP induction in both groups (E), but this decrease was significantly more pronounced in the CASPR2 group (F). Representative EEG four of the CASPR2-mAb infused animals, 1) a hyper-excitability event seen on day 5 during the night (Supplemental video 1), 2,3,4) and when awake (G). Mean hourly 1-second ictal events seen over the total period of infusion, seen here for CASPR2 (n = 6) and control (n = 5) (H). Comparison of hourly measurements of coastline length between control (n = 5) and Caspr2 mAb (n = 6) infused rats over 21 day recording period (P≤0.0001; Mann-Whitney). Bars indicate mean ± SEM (I). Comparison of hourly EEG power averages in frequency bands shown between control (n = 5) and CASPR2 mAb (n = 6) infused rats over 21 day recording period (P≤0.0001; Mann-Whitney). Bars indicate mean ± SEM (J).

Fig. 4

In vivo effects of infusion of CASPR2 reactive monoclonal antibody in mice. Representative tile scan image of coronal section of mouse infused with CASPR2 antibody or control after 14 days, with human IgG shown in green (A). Mean fluorescence intensity as measured in 10 areas, in 3 coronal slices of each animal on both the ipsi and contra lateral side to the injection site, for each animal (n = 6 for each group, p < 0.00001; T test) (B). Time spent at each stranger mouse ‘S1, S2′, with S1 serving as the familiar mouse in the novelty test, or empty cage ‘E’ after 10 days of infusion of either control or CASPR2 antibody in a three-chamber test paradigm (n = 14 for each group; p < 0.01; paired t-test) (C). Other behavioral paradigms tested can be found in supplemental Fig. 2. Functional connectivity group comparisons (CASPR2 > control, n = 14 each group) through assessed using dual regression as are shown superimposed on coronal sections of the Allen Mouse Brain Atlas (AMBA), with neo represented by coronal anatomical MRI scan image with cortex shown in light blue, hippocampus in green, thalamus area in red and mesencephalon in grey; ICA components in yellow–red; and significant group differences in blue-light blue. Extracted individual animal functional connectivity values are shown separately for both groups in scatter plots, in corresponding positions to results on AMBA sections (p < 0.001; T test; D). DTI analysis using the following predetermined clusters; 1) amygdala, 2) substantia nigra, 3) olfactory bulb, 4) periaqueductal gray, 5) trigeminal pontine nucleus, 6) trigeminal spinal nucleus, 7) trigeminal sensory nucleus, 8) superior olivary complex, 9) inferior olivary complex.Affected connections shown in a representative 3D model of a mouse brain, comparison represents CASPR2 vs Control (n = 14 each group), with the difference in CS shown in gradient (E). DTI cluster size analysis CASPR2 vs Control (n = 14 each group) with the difference shown in gradient within coronal, transversal, and sagittal cross section of a representative anatomical mouse scan (F). Brain area volume as measured by T2 anatomical scan for amygdala, hippocampus, caudatoputamen, thalamus and hypothalamus, after 14 days of infusion (n = 14 per group) (G).