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

Pinatel D, Hivert B, Boucraut J, Saint-Martin M, Rogemond V, Zoupi L, Karagogeos D, Honnorat J, Faivre-Sarrailh C - Front Cell Neurosci (2015)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Aix Marseille Université, CNRS, Centre de Recherche en Neurobiologie et Neurophysiologie de Marseille, CRN2M-UMR7286, Faculté de Médecine Nord Marseille, France.

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.

No MeSH data available.


Related in: MedlinePlus

Epitope mapping of anti-Caspr2 autoantibodies in LE patients. (A) Caspr2-HA constructs encompassing sequential deletions: Δ1 deleted of the N-terminal discoïdin and Laminin-G1 domains, Δ2 deleted of the Laminin-G2 and EGF-like1 domains, Δ3 deleted of the central fibrinogen and Laminin-G3 domains, Δ4 deleted of the EGF-like2, and Laminin-G4 domains, Caspr2-Disc-LNG1, Caspr2-Disc, and Caspr2-LNG1 only including the Discoïdin and/or LamininG1 modules in the ectodomain. (B) Flow cytometry analysis of anti-Caspr2 IgG titer of LE1 serum. Untransfected and Caspr2-HA-transfected HEK cells were incubated with serial dilutions of LE1 serum (1/3,200–1/51,200) and phycoerythrin-conjugated anti-human IgG (Anti-IgG-PE). (C) Flow cytometry analysis of anti-Caspr2 serum IgGs of LE1 and LE6 on HEK cells transfected with full-length or Δ1 deleted Caspr2-HA construct.
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Figure 2: Epitope mapping of anti-Caspr2 autoantibodies in LE patients. (A) Caspr2-HA constructs encompassing sequential deletions: Δ1 deleted of the N-terminal discoïdin and Laminin-G1 domains, Δ2 deleted of the Laminin-G2 and EGF-like1 domains, Δ3 deleted of the central fibrinogen and Laminin-G3 domains, Δ4 deleted of the EGF-like2, and Laminin-G4 domains, Caspr2-Disc-LNG1, Caspr2-Disc, and Caspr2-LNG1 only including the Discoïdin and/or LamininG1 modules in the ectodomain. (B) Flow cytometry analysis of anti-Caspr2 IgG titer of LE1 serum. Untransfected and Caspr2-HA-transfected HEK cells were incubated with serial dilutions of LE1 serum (1/3,200–1/51,200) and phycoerythrin-conjugated anti-human IgG (Anti-IgG-PE). (C) Flow cytometry analysis of anti-Caspr2 serum IgGs of LE1 and LE6 on HEK cells transfected with full-length or Δ1 deleted Caspr2-HA construct.

Mentions: The pCDNA3-Caspr2-HA construct encodes human Caspr2 with the HA epitope inserted downstream the signal peptide between the residues Trp26 and Thr27 (Bel et al., 2009). Caspr2-mcherry was generated by PCR amplification and insertion into the EcoR1-BamH1 sites of pmCherry-N1 vector. The Caspr2-HA deleted constructs, Caspr2Δ1 (Δ32-361), Caspr2Δ2 (Δ362-600), Caspr2Δ3 (Δ600-950), Caspr2Δ4 (Δ955–1169) were generated by QuickChange mutagenesis (Agilent Technologies). The Caspr2-Discoïdin-LamininG1, Caspr2-Discoïdin, Caspr2-LamininG1 (Figure 2A) were obtained using reverse PCR on HA-tagged full length Caspr2 plasmid and the fragment fusion was performed using the In-Fusion kit (Clontech). The Caspr2-Fc construct was generated by PCR amplification of Caspr2 extracellular domain (amino acids 1–1242) and insertion into the Kpn1-Not1 cloning sites of pIg-plus vector. The human TAG-1-GFP construct was generated by inserting GFP downstream the signal peptide. PCR amplified products were verified by sequencing (Beckman Coulter Genomics). Plasmids encoding human LGI1, ADAM22, ADAM23 were purchased from Origene. Gephyrin-GFP is a kind gift of Dr. F. Ango.


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

Pinatel D, Hivert B, Boucraut J, Saint-Martin M, Rogemond V, Zoupi L, Karagogeos D, Honnorat J, Faivre-Sarrailh C - Front Cell Neurosci (2015)

Epitope mapping of anti-Caspr2 autoantibodies in LE patients. (A) Caspr2-HA constructs encompassing sequential deletions: Δ1 deleted of the N-terminal discoïdin and Laminin-G1 domains, Δ2 deleted of the Laminin-G2 and EGF-like1 domains, Δ3 deleted of the central fibrinogen and Laminin-G3 domains, Δ4 deleted of the EGF-like2, and Laminin-G4 domains, Caspr2-Disc-LNG1, Caspr2-Disc, and Caspr2-LNG1 only including the Discoïdin and/or LamininG1 modules in the ectodomain. (B) Flow cytometry analysis of anti-Caspr2 IgG titer of LE1 serum. Untransfected and Caspr2-HA-transfected HEK cells were incubated with serial dilutions of LE1 serum (1/3,200–1/51,200) and phycoerythrin-conjugated anti-human IgG (Anti-IgG-PE). (C) Flow cytometry analysis of anti-Caspr2 serum IgGs of LE1 and LE6 on HEK cells transfected with full-length or Δ1 deleted Caspr2-HA construct.
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Related In: Results  -  Collection

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Figure 2: Epitope mapping of anti-Caspr2 autoantibodies in LE patients. (A) Caspr2-HA constructs encompassing sequential deletions: Δ1 deleted of the N-terminal discoïdin and Laminin-G1 domains, Δ2 deleted of the Laminin-G2 and EGF-like1 domains, Δ3 deleted of the central fibrinogen and Laminin-G3 domains, Δ4 deleted of the EGF-like2, and Laminin-G4 domains, Caspr2-Disc-LNG1, Caspr2-Disc, and Caspr2-LNG1 only including the Discoïdin and/or LamininG1 modules in the ectodomain. (B) Flow cytometry analysis of anti-Caspr2 IgG titer of LE1 serum. Untransfected and Caspr2-HA-transfected HEK cells were incubated with serial dilutions of LE1 serum (1/3,200–1/51,200) and phycoerythrin-conjugated anti-human IgG (Anti-IgG-PE). (C) Flow cytometry analysis of anti-Caspr2 serum IgGs of LE1 and LE6 on HEK cells transfected with full-length or Δ1 deleted Caspr2-HA construct.
Mentions: The pCDNA3-Caspr2-HA construct encodes human Caspr2 with the HA epitope inserted downstream the signal peptide between the residues Trp26 and Thr27 (Bel et al., 2009). Caspr2-mcherry was generated by PCR amplification and insertion into the EcoR1-BamH1 sites of pmCherry-N1 vector. The Caspr2-HA deleted constructs, Caspr2Δ1 (Δ32-361), Caspr2Δ2 (Δ362-600), Caspr2Δ3 (Δ600-950), Caspr2Δ4 (Δ955–1169) were generated by QuickChange mutagenesis (Agilent Technologies). The Caspr2-Discoïdin-LamininG1, Caspr2-Discoïdin, Caspr2-LamininG1 (Figure 2A) were obtained using reverse PCR on HA-tagged full length Caspr2 plasmid and the fragment fusion was performed using the In-Fusion kit (Clontech). The Caspr2-Fc construct was generated by PCR amplification of Caspr2 extracellular domain (amino acids 1–1242) and insertion into the Kpn1-Not1 cloning sites of pIg-plus vector. The human TAG-1-GFP construct was generated by inserting GFP downstream the signal peptide. PCR amplified products were verified by sequencing (Beckman Coulter Genomics). Plasmids encoding human LGI1, ADAM22, ADAM23 were purchased from Origene. Gephyrin-GFP is a kind gift of Dr. F. Ango.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Aix Marseille Université, CNRS, Centre de Recherche en Neurobiologie et Neurophysiologie de Marseille, CRN2M-UMR7286, Faculté de Médecine Nord Marseille, France.

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.

No MeSH data available.


Related in: MedlinePlus