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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

TAG-1 is required for Caspr2-Fc binding on hippocampal neurons. (A) N2a neuroblastoma cells were transfected with TAG-1-GFP (green) and incubated with preclustered Caspr2-Fc (red). Caspr2-Fc only bound on TAG-1-GFP expressing N2a cells. (B–G) DIV8 hippocampal neurons were incubated with pre-clustered Caspr2-Fc (red) and cells were fixed and permeabilized before immunostaining for MAP2 (blue). Wild-type neurons were untransfected (B) or transfected with TAG-1-GFP (C), or double-transfected with LGI1-GFP and ADAM22 (F) or LGI1-GFP and ADAM23 (G). Caspr2-Fc strongly bound the TAG-1-GFP-expressing neuron (green) by comparison with untransfected neurons (C). Transfection of LGI-GFP and ADAM22 or ADAM23 had no effect. (D,E) DIV8 hippocampal neurons from Tag-1-/- mice were untransfected (D) or transfected with TAG-1-GFP (E). Caspr2-Fc strongly bound the TAG-1-GFP-expressing neuron (green) and did not bind untransfected Tag1-/- neurons. (H,H’) DIV14 hippocampal neurons were co-transfected with TAG-1-GFP and mCherry. At DIV17, neurons were surface labeled with anti-GFP antibodies, fixed, and permeabilized before immunostaining for Synaptophysin (blue). Note that TAG-1-GFP clusters indicated with arrowheads on the shaft (H) or spines (H’) were facing Synaptophysin presynaptic sites. (A–G) Single confocal sections. (H,H’) z-stacks of four confocal sections with z-step of 0.5 μm Bars: 10 μm, in (A–G), 4 μm in (H,H’).
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Figure 8: TAG-1 is required for Caspr2-Fc binding on hippocampal neurons. (A) N2a neuroblastoma cells were transfected with TAG-1-GFP (green) and incubated with preclustered Caspr2-Fc (red). Caspr2-Fc only bound on TAG-1-GFP expressing N2a cells. (B–G) DIV8 hippocampal neurons were incubated with pre-clustered Caspr2-Fc (red) and cells were fixed and permeabilized before immunostaining for MAP2 (blue). Wild-type neurons were untransfected (B) or transfected with TAG-1-GFP (C), or double-transfected with LGI1-GFP and ADAM22 (F) or LGI1-GFP and ADAM23 (G). Caspr2-Fc strongly bound the TAG-1-GFP-expressing neuron (green) by comparison with untransfected neurons (C). Transfection of LGI-GFP and ADAM22 or ADAM23 had no effect. (D,E) DIV8 hippocampal neurons from Tag-1-/- mice were untransfected (D) or transfected with TAG-1-GFP (E). Caspr2-Fc strongly bound the TAG-1-GFP-expressing neuron (green) and did not bind untransfected Tag1-/- neurons. (H,H’) DIV14 hippocampal neurons were co-transfected with TAG-1-GFP and mCherry. At DIV17, neurons were surface labeled with anti-GFP antibodies, fixed, and permeabilized before immunostaining for Synaptophysin (blue). Note that TAG-1-GFP clusters indicated with arrowheads on the shaft (H) or spines (H’) were facing Synaptophysin presynaptic sites. (A–G) Single confocal sections. (H,H’) z-stacks of four confocal sections with z-step of 0.5 μm Bars: 10 μm, in (A–G), 4 μm in (H,H’).

Mentions: Contactin 2/TAG-1 is an Ig-CAM that associates with Caspr2 and the VGKC complex at juxtaparanodes (Poliak et al., 2003; Traka et al., 2003). It was previously reported that TAG-1 strongly associates in cis with Caspr2 whereas the trans-interaction remains elusive, since TAG-1-Fc does not bind to Caspr2 expressed at the cell membrane of HEK cells (Traka et al., 2003). Conversely, we observed that the pre-clustered Caspr2-Fc chimera bound N2a cells transfected with GPI-anchored TAG-1 fused with GFP downstream the signal peptide (Figure 8A). In addition, Caspr2-Fc binding was strongly enhanced on neurons transfected with TAG-1 when compared with untransfected neurons (Figures 8B,C), indicating that the trans-interaction with TAG-1 strongly occurs in neuronal cells. Next, we tested whether Caspr2-Fc bound on hippocampal neurons from Tag-1-/- mice. Caspr2-Fc binding was faintly detected on TAG-1-deficient neurons (Figure 8D). In contrast, Caspr2-Fc strongly labeled hippocampal neurons from Tag-1-/- mice that were transfected with TAG-1-GFP in contrast with untransfected neurons (Figure 8E).


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)

TAG-1 is required for Caspr2-Fc binding on hippocampal neurons. (A) N2a neuroblastoma cells were transfected with TAG-1-GFP (green) and incubated with preclustered Caspr2-Fc (red). Caspr2-Fc only bound on TAG-1-GFP expressing N2a cells. (B–G) DIV8 hippocampal neurons were incubated with pre-clustered Caspr2-Fc (red) and cells were fixed and permeabilized before immunostaining for MAP2 (blue). Wild-type neurons were untransfected (B) or transfected with TAG-1-GFP (C), or double-transfected with LGI1-GFP and ADAM22 (F) or LGI1-GFP and ADAM23 (G). Caspr2-Fc strongly bound the TAG-1-GFP-expressing neuron (green) by comparison with untransfected neurons (C). Transfection of LGI-GFP and ADAM22 or ADAM23 had no effect. (D,E) DIV8 hippocampal neurons from Tag-1-/- mice were untransfected (D) or transfected with TAG-1-GFP (E). Caspr2-Fc strongly bound the TAG-1-GFP-expressing neuron (green) and did not bind untransfected Tag1-/- neurons. (H,H’) DIV14 hippocampal neurons were co-transfected with TAG-1-GFP and mCherry. At DIV17, neurons were surface labeled with anti-GFP antibodies, fixed, and permeabilized before immunostaining for Synaptophysin (blue). Note that TAG-1-GFP clusters indicated with arrowheads on the shaft (H) or spines (H’) were facing Synaptophysin presynaptic sites. (A–G) Single confocal sections. (H,H’) z-stacks of four confocal sections with z-step of 0.5 μm Bars: 10 μm, in (A–G), 4 μm in (H,H’).
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Figure 8: TAG-1 is required for Caspr2-Fc binding on hippocampal neurons. (A) N2a neuroblastoma cells were transfected with TAG-1-GFP (green) and incubated with preclustered Caspr2-Fc (red). Caspr2-Fc only bound on TAG-1-GFP expressing N2a cells. (B–G) DIV8 hippocampal neurons were incubated with pre-clustered Caspr2-Fc (red) and cells were fixed and permeabilized before immunostaining for MAP2 (blue). Wild-type neurons were untransfected (B) or transfected with TAG-1-GFP (C), or double-transfected with LGI1-GFP and ADAM22 (F) or LGI1-GFP and ADAM23 (G). Caspr2-Fc strongly bound the TAG-1-GFP-expressing neuron (green) by comparison with untransfected neurons (C). Transfection of LGI-GFP and ADAM22 or ADAM23 had no effect. (D,E) DIV8 hippocampal neurons from Tag-1-/- mice were untransfected (D) or transfected with TAG-1-GFP (E). Caspr2-Fc strongly bound the TAG-1-GFP-expressing neuron (green) and did not bind untransfected Tag1-/- neurons. (H,H’) DIV14 hippocampal neurons were co-transfected with TAG-1-GFP and mCherry. At DIV17, neurons were surface labeled with anti-GFP antibodies, fixed, and permeabilized before immunostaining for Synaptophysin (blue). Note that TAG-1-GFP clusters indicated with arrowheads on the shaft (H) or spines (H’) were facing Synaptophysin presynaptic sites. (A–G) Single confocal sections. (H,H’) z-stacks of four confocal sections with z-step of 0.5 μm Bars: 10 μm, in (A–G), 4 μm in (H,H’).
Mentions: Contactin 2/TAG-1 is an Ig-CAM that associates with Caspr2 and the VGKC complex at juxtaparanodes (Poliak et al., 2003; Traka et al., 2003). It was previously reported that TAG-1 strongly associates in cis with Caspr2 whereas the trans-interaction remains elusive, since TAG-1-Fc does not bind to Caspr2 expressed at the cell membrane of HEK cells (Traka et al., 2003). Conversely, we observed that the pre-clustered Caspr2-Fc chimera bound N2a cells transfected with GPI-anchored TAG-1 fused with GFP downstream the signal peptide (Figure 8A). In addition, Caspr2-Fc binding was strongly enhanced on neurons transfected with TAG-1 when compared with untransfected neurons (Figures 8B,C), indicating that the trans-interaction with TAG-1 strongly occurs in neuronal cells. Next, we tested whether Caspr2-Fc bound on hippocampal neurons from Tag-1-/- mice. Caspr2-Fc binding was faintly detected on TAG-1-deficient neurons (Figure 8D). In contrast, Caspr2-Fc strongly labeled hippocampal neurons from Tag-1-/- mice that were transfected with TAG-1-GFP in contrast with untransfected neurons (Figure 8E).

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