Inhibitory axons are targeted in hippocampal cell culture by anti-Caspr2 autoantibodies associated with limbic encephalitis

Abstract

Contactin-associated protein-like 2 (Caspr2), also known as CNTNAP2, is a cell adhesion molecule that clusters voltage-gated potassium channels (Kv1.1/1.2) at the juxtaparanodes of myelinated axons and may regulate axonal excitability. As a component of the Kv1 complex, Caspr2 has been identified as a target in neuromyotonia and Morvan syndrome, but also in some cases of autoimmune limbic encephalitis (LE). How anti-Caspr2 autoimmunity is linked with the central neurological symptoms is still elusive. In the present study, using anti-Caspr2 antibodies from seven patients affected by pure LE, we determined that IgGs in the cerebrospinal fluid of four out seven patients were selectively directed against the N-terminal Discoïdin and LamininG1 modules of Caspr2. Using live immunolabeling of cultured hippocampal neurons, we determined that serum IgGs in all patients strongly targeted inhibitory interneurons. Caspr2 was highly detected on GAD65-positive axons that are surrounding the cell bodies and at the VGAT-positive inhibitory presynaptic contacts. Functional assays indicated that LE autoantibodies may induce alteration of Gephyrin clusters at inhibitory synaptic contacts. Next, we generated a Caspr2-Fc chimera to reveal Caspr2 receptors on hippocampal neurons localized at the somato-dendritic compartment and post-synapse. Caspr2-Fc binding was strongly increased on TAG-1-transfected neurons and conversely, Caspr2-Fc did not bind hippocampal neurons from TAG-1-deficient mice. Our data indicate that Caspr2 may participate as a cell recognition molecule in the dynamics of inhibitory networks. This study provides new insight into the potential pathogenic effect of anti-Caspr2 autoantibodies in central hyperexcitability that may be related with perturbation of inhibitory interneuron activity.

The Kv1-associated molecules TAG-1 and Caspr2 are selectively targeted to the axon initial segment in hippocampal neurons

“…The mouse anti-TAG-1 1C12 mAb and the rabbit anti-Caspr2 antiserum were as previously described (Bel et al, 2009;Traka et al, 2003). Anti-Caspr2 antibodies from limbic encephalitis patients were characterized previously (Pinatel et al, 2015). Alexa Fluor 488-, 568-and 647-conjugated secondary antibodies were obtained from Molecular Probes (ThermoFisher).…”

“…Nr-Caspr2cyt constructs with deletions of the binding site for 4.1B (Δ1288-1305), the PDZ-binding domain (stop at residue 1330) or the C-terminal region (stop codon at residue 1306) were generated. The human TAG-1-GFP and Caspr2-GFP constructs with GFP downstream of the signal peptide were as described previously (Bel et al, 2009;Pinatel et al, 2015). The human TAG-1-GFP deletion constructs were generated by PCR amplification from the previously described TAG-1-Ig and TAG-1-Fn constructs (Tzimourakas et al, 2007), and were inserted in the XhoI/ HindIII sites of a pEGFP-C1 plasmid vector modified to contain the signal peptide of TAG-1 upstream of GFP.…”

“…The Caspr2-HA deletion constructs, Caspr2Δ1 (Δ32-361), Caspr2Δ2 (Δ362-600), Caspr2Δ3 (Δ600-950), Caspr2Δ4 (Δ955-1169) were as described previously (Pinatel et al, 2015). The NrCAM-Caspr2cyt construct was generated by insertion of the Caspr2 transmembrane and cytoplasmic regions downstream the signal peptide and Ig domains of NrCAM, and is tagged with HA and GFP (Falk et al, 2004).…”

“…We observed that Caspr2 and TAG-1 were differentially distributed. As previously reported (Pinatel et al, 2015), we used anti-Caspr2 auto-antibodies from patients with limbic encephalitis to show that Caspr2 surface labeling was polarized to axons (Fig. 1A, red), but not enriched at the AIS, which can be labeled for ankyrinG (Fig.…”

“…We analyzed the distribution of a series of HA-tagged Caspr2 constructs with sequential deletions of modules in the ectodomain ( Fig. 3A; Pinatel et al, 2015). After transfection at DIV7, hippocampal neurons were surface labeled for HA (green), and then fixed and permeabilized before staining for ankyrinG as an AIS marker (blue).…”