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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

GAD65-positive neurons are the main target of anti-Caspr2 LE1 autoantibodies. DIV4 hippocampal neurons were surface labeled with LE1 IgGs (red), fixed and permeabilized before immunostaining for MAP2 (blue) as a neuronal marker and GAD65 (green) to identify inhibitory neurons. (A) Low magnification image showing neurons that were surface labeled for Caspr2 (red). Most of them were GAD65-positive (green, arrowheads) and few were GAD65-negative (arrows). The box enlarged in C shows GAD65-positive neurons in green that were surface labeled for Caspr2. The arrowhead points to an isolated inhibitory neuron strongly labeled for Caspr2 on the soma (B) Quantitative analysis of the percentage of total neurons, GAD65-positive and GAD65-negative neurons that were surface labeled for Caspr2. Means ± SEM of three independent experiments. (A) Tiling of 5 × 4 confocal images acquired with the 63x objective, z-stack of 6 confocal sections with z-step of 0.5 μm. Bar is in A, 80 μm; in (C), 35 μm.
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Figure 3: GAD65-positive neurons are the main target of anti-Caspr2 LE1 autoantibodies. DIV4 hippocampal neurons were surface labeled with LE1 IgGs (red), fixed and permeabilized before immunostaining for MAP2 (blue) as a neuronal marker and GAD65 (green) to identify inhibitory neurons. (A) Low magnification image showing neurons that were surface labeled for Caspr2 (red). Most of them were GAD65-positive (green, arrowheads) and few were GAD65-negative (arrows). The box enlarged in C shows GAD65-positive neurons in green that were surface labeled for Caspr2. The arrowhead points to an isolated inhibitory neuron strongly labeled for Caspr2 on the soma (B) Quantitative analysis of the percentage of total neurons, GAD65-positive and GAD65-negative neurons that were surface labeled for Caspr2. Means ± SEM of three independent experiments. (A) Tiling of 5 × 4 confocal images acquired with the 63x objective, z-stack of 6 confocal sections with z-step of 0.5 μm. Bar is in A, 80 μm; in (C), 35 μm.

Mentions: Since only a subpopulation of axons was labeled using anti-Caspr2 LE1 serum IgGs, we further investigated whether excitatory or inhibitory subpopulation of neurons may be differentially targeted. DIV4 neurons were analyzed to study the somato-dendritic expression of Caspr2 at early stage. We first determined that 22% of MAP2-positive neurons were GABAergic using glutamate decarboxylase GAD65 as a marker (Figures 3A,B). Next using immunolabeling with LE1 serum IgGs, we estimated that 58% of the inhibitory neurons in contrast to only 4% of excitatory neurons (negative for GAD65, arrows) expressed Caspr2 at DIV4 (Figures 3A,B; Table 3). Thus, most of the Caspr2-positive neurons (81%) were inhibitory neurons as illustrated in Figure 3C. In the same manner, quantitative analyses were performed using LE2–LE5 serum IgGs and indicated that 51–68% of GAD65-positive neurons and only 4–8% of GAD65-negative neurons were targeted by these patients IgGs (Table 3).


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)

GAD65-positive neurons are the main target of anti-Caspr2 LE1 autoantibodies. DIV4 hippocampal neurons were surface labeled with LE1 IgGs (red), fixed and permeabilized before immunostaining for MAP2 (blue) as a neuronal marker and GAD65 (green) to identify inhibitory neurons. (A) Low magnification image showing neurons that were surface labeled for Caspr2 (red). Most of them were GAD65-positive (green, arrowheads) and few were GAD65-negative (arrows). The box enlarged in C shows GAD65-positive neurons in green that were surface labeled for Caspr2. The arrowhead points to an isolated inhibitory neuron strongly labeled for Caspr2 on the soma (B) Quantitative analysis of the percentage of total neurons, GAD65-positive and GAD65-negative neurons that were surface labeled for Caspr2. Means ± SEM of three independent experiments. (A) Tiling of 5 × 4 confocal images acquired with the 63x objective, z-stack of 6 confocal sections with z-step of 0.5 μm. Bar is in A, 80 μm; in (C), 35 μm.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
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Figure 3: GAD65-positive neurons are the main target of anti-Caspr2 LE1 autoantibodies. DIV4 hippocampal neurons were surface labeled with LE1 IgGs (red), fixed and permeabilized before immunostaining for MAP2 (blue) as a neuronal marker and GAD65 (green) to identify inhibitory neurons. (A) Low magnification image showing neurons that were surface labeled for Caspr2 (red). Most of them were GAD65-positive (green, arrowheads) and few were GAD65-negative (arrows). The box enlarged in C shows GAD65-positive neurons in green that were surface labeled for Caspr2. The arrowhead points to an isolated inhibitory neuron strongly labeled for Caspr2 on the soma (B) Quantitative analysis of the percentage of total neurons, GAD65-positive and GAD65-negative neurons that were surface labeled for Caspr2. Means ± SEM of three independent experiments. (A) Tiling of 5 × 4 confocal images acquired with the 63x objective, z-stack of 6 confocal sections with z-step of 0.5 μm. Bar is in A, 80 μm; in (C), 35 μm.
Mentions: Since only a subpopulation of axons was labeled using anti-Caspr2 LE1 serum IgGs, we further investigated whether excitatory or inhibitory subpopulation of neurons may be differentially targeted. DIV4 neurons were analyzed to study the somato-dendritic expression of Caspr2 at early stage. We first determined that 22% of MAP2-positive neurons were GABAergic using glutamate decarboxylase GAD65 as a marker (Figures 3A,B). Next using immunolabeling with LE1 serum IgGs, we estimated that 58% of the inhibitory neurons in contrast to only 4% of excitatory neurons (negative for GAD65, arrows) expressed Caspr2 at DIV4 (Figures 3A,B; Table 3). Thus, most of the Caspr2-positive neurons (81%) were inhibitory neurons as illustrated in Figure 3C. In the same manner, quantitative analyses were performed using LE2–LE5 serum IgGs and indicated that 51–68% of GAD65-positive neurons and only 4–8% of GAD65-negative neurons were targeted by these patients IgGs (Table 3).

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