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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.


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The Caspr2-Fc binding sites are localized at post-synaptic contacts. (A) Hippocampal neurons were transfected at DIV14 with GFP and incubated at DIV21 with preclustered Caspr2-Fc (red). Cells were fixed, permeabilized and immunostained for Synaptophysin (blue). (A’,A”) Insets show the distribution of Caspr2-Fc binding sites (red) on the dendritic shaft (arrowheads) or on spines (arrow) that were contacting Synaptophysin pre-synaptic sites (blue). The green channel is turned down in (A”) to visualize Synaptophysin clusters. (B) Quantitative analysis of the number of post-synaptic sites bound with Caspr2-Fc on spines and shaft. Means ± SEM, n = 21 dendrites of seven neurons. Single confocal sections. Bar is in (A,B), 20 μm; in insets, 4 μm.
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Figure 7: The Caspr2-Fc binding sites are localized at post-synaptic contacts. (A) Hippocampal neurons were transfected at DIV14 with GFP and incubated at DIV21 with preclustered Caspr2-Fc (red). Cells were fixed, permeabilized and immunostained for Synaptophysin (blue). (A’,A”) Insets show the distribution of Caspr2-Fc binding sites (red) on the dendritic shaft (arrowheads) or on spines (arrow) that were contacting Synaptophysin pre-synaptic sites (blue). The green channel is turned down in (A”) to visualize Synaptophysin clusters. (B) Quantitative analysis of the number of post-synaptic sites bound with Caspr2-Fc on spines and shaft. Means ± SEM, n = 21 dendrites of seven neurons. Single confocal sections. Bar is in (A,B), 20 μm; in insets, 4 μm.

Mentions: Next, we analyzed whether the Caspr2 binding sites may be distributed at the post-synaptic sites. Neurons were transfected with GFP at DIV14 to clearly visualize dendrites and spines of isolated neurons at DIV21 (Figure 7A). Synaptic contacts on dendritic shafts (arrowheads) or spines (arrow) were detected using Synaptophysin as a pre-synaptic marker. As shown in high magnification pictures, Caspr2 binding sites (red) were detected on shafts at the contact with the pre-synaptic marker (blue; Figures 7A’,A”). Quantitative analysis indicated that 58% of post-synapses on shafts and 48% on spines were labeled with Caspr2-Fc chimera (n = 21 images; Figure 7B). Thus, the receptors of Caspr2 were present both at inhibitory and excitatory post-synaptic sites on shafts and spines, respectively. The Caspr2-binding sites at the inhibitory post-synapses were complementary to the distribution of Caspr2 at inhibitory pre-synaptic sites.


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)

The Caspr2-Fc binding sites are localized at post-synaptic contacts. (A) Hippocampal neurons were transfected at DIV14 with GFP and incubated at DIV21 with preclustered Caspr2-Fc (red). Cells were fixed, permeabilized and immunostained for Synaptophysin (blue). (A’,A”) Insets show the distribution of Caspr2-Fc binding sites (red) on the dendritic shaft (arrowheads) or on spines (arrow) that were contacting Synaptophysin pre-synaptic sites (blue). The green channel is turned down in (A”) to visualize Synaptophysin clusters. (B) Quantitative analysis of the number of post-synaptic sites bound with Caspr2-Fc on spines and shaft. Means ± SEM, n = 21 dendrites of seven neurons. Single confocal sections. Bar is in (A,B), 20 μm; in insets, 4 μm.
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Related In: Results  -  Collection

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Figure 7: The Caspr2-Fc binding sites are localized at post-synaptic contacts. (A) Hippocampal neurons were transfected at DIV14 with GFP and incubated at DIV21 with preclustered Caspr2-Fc (red). Cells were fixed, permeabilized and immunostained for Synaptophysin (blue). (A’,A”) Insets show the distribution of Caspr2-Fc binding sites (red) on the dendritic shaft (arrowheads) or on spines (arrow) that were contacting Synaptophysin pre-synaptic sites (blue). The green channel is turned down in (A”) to visualize Synaptophysin clusters. (B) Quantitative analysis of the number of post-synaptic sites bound with Caspr2-Fc on spines and shaft. Means ± SEM, n = 21 dendrites of seven neurons. Single confocal sections. Bar is in (A,B), 20 μm; in insets, 4 μm.
Mentions: Next, we analyzed whether the Caspr2 binding sites may be distributed at the post-synaptic sites. Neurons were transfected with GFP at DIV14 to clearly visualize dendrites and spines of isolated neurons at DIV21 (Figure 7A). Synaptic contacts on dendritic shafts (arrowheads) or spines (arrow) were detected using Synaptophysin as a pre-synaptic marker. As shown in high magnification pictures, Caspr2 binding sites (red) were detected on shafts at the contact with the pre-synaptic marker (blue; Figures 7A’,A”). Quantitative analysis indicated that 58% of post-synapses on shafts and 48% on spines were labeled with Caspr2-Fc chimera (n = 21 images; Figure 7B). Thus, the receptors of Caspr2 were present both at inhibitory and excitatory post-synaptic sites on shafts and spines, respectively. The Caspr2-binding sites at the inhibitory post-synapses were complementary to the distribution of Caspr2 at inhibitory pre-synaptic sites.

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