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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

The binding sites of Caspr2-Fc are localized on the somato-dendritic compartment. Hippocampal neurons at DIV4 (A–C) and DIV7 (D,E) were incubated with 10 μg/ml Caspr2-Fc preclustered with Alexa-conjugated anti-Fc IgGs for 30 min at 37°C. (A,B) DIV4 neurons bound with Caspr2-Fc (red). Cells were fixed and permeabilized before double-staining for MAP2 (blue) and GAD65 (green). (A,B) show representative images of GAD65-negative neurons (white arrow, A) and GAD65-positive neurons (green arrow, B) labeled with Caspr2-Fc. (C) Quantitative analysis of the percentage of total neurons, GAD65-positive and GAD65-negative neurons that were surface labeled for Caspr2-Fc. Means ± SEM of three independent experiments, n = 504 neurons. (D,E) DIV7 neurons bound with Caspr2-Fc (green) were double-stained for MAP2 (D, blue) or tau (E, red). The insets in (D’,E’) show that Caspr2-Fc preferentially bound on MAP2-positive dendrites and not on tau-positive axons. (A,B)z-stacks of six confocal sections with z-step of 0.5 μm. (D,E) Single optical sections of confocal images. Bar is in (A,B), 20 μm; in (D,E), 10 μm; in insets, 7 μm.
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Figure 6: The binding sites of Caspr2-Fc are localized on the somato-dendritic compartment. Hippocampal neurons at DIV4 (A–C) and DIV7 (D,E) were incubated with 10 μg/ml Caspr2-Fc preclustered with Alexa-conjugated anti-Fc IgGs for 30 min at 37°C. (A,B) DIV4 neurons bound with Caspr2-Fc (red). Cells were fixed and permeabilized before double-staining for MAP2 (blue) and GAD65 (green). (A,B) show representative images of GAD65-negative neurons (white arrow, A) and GAD65-positive neurons (green arrow, B) labeled with Caspr2-Fc. (C) Quantitative analysis of the percentage of total neurons, GAD65-positive and GAD65-negative neurons that were surface labeled for Caspr2-Fc. Means ± SEM of three independent experiments, n = 504 neurons. (D,E) DIV7 neurons bound with Caspr2-Fc (green) were double-stained for MAP2 (D, blue) or tau (E, red). The insets in (D’,E’) show that Caspr2-Fc preferentially bound on MAP2-positive dendrites and not on tau-positive axons. (A,B)z-stacks of six confocal sections with z-step of 0.5 μm. (D,E) Single optical sections of confocal images. Bar is in (A,B), 20 μm; in (D,E), 10 μm; in insets, 7 μm.

Mentions: Caspr2 belongs to the family of neurexins, which are pre-synaptic CAMs. Studies in culture indicate that neurexins are implicated in synaptogenesis by inducing the clustering of post-synaptic neuroligins (Dean et al., 2003; Craig and Kang, 2007). Neurexin/neuroligin association promotes the formation of excitatory and inhibitory synapses by interacting with PSD95 or Gephyrin, respectively. We asked whether Caspr2 might be also involved in trans-synaptic contacts. With this aim, we generated a Caspr2-Fc chimera to detect Caspr2 binding sites in hippocampal neuronal culture. Caspr2-Fc plasmid was transfected in HEK cells and the recombinant protein purified from the culture supernatant using Protein A-affinity chromatography. The chimera pre-clustered with fluorescent anti-Fc IgG was incubated with hippocampal neurons at DIV4 (Figure 6). We observed that Caspr2 binding sites were present on both GAD65-negative (arrow in Figure 6A) and GAD65-positive neurons (green arrow in Figure 6B). Quantitative analysis indicated that Caspr2-Fc bound 36% of the total neurons (Figure 6C). Caspr2-Fc bound to the somato-dendritic compartment of DIV7 neurons as determined using double-staining for MAP2 (Figures 6D,D’). In contrast, Caspr2-Fc was not co-localized with axons immunostained with anti-tau mAb (Figures 6E,E’). Caspr2-Fc binding sites were distributed on the somato-dendritic compartment of both inhibitory (26 ± 6%) and excitatory (38 ± 5%) neurons as analyzed at DIV4 (n = 504 neurons, three coverslips; Figure 6C).


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 binding sites of Caspr2-Fc are localized on the somato-dendritic compartment. Hippocampal neurons at DIV4 (A–C) and DIV7 (D,E) were incubated with 10 μg/ml Caspr2-Fc preclustered with Alexa-conjugated anti-Fc IgGs for 30 min at 37°C. (A,B) DIV4 neurons bound with Caspr2-Fc (red). Cells were fixed and permeabilized before double-staining for MAP2 (blue) and GAD65 (green). (A,B) show representative images of GAD65-negative neurons (white arrow, A) and GAD65-positive neurons (green arrow, B) labeled with Caspr2-Fc. (C) Quantitative analysis of the percentage of total neurons, GAD65-positive and GAD65-negative neurons that were surface labeled for Caspr2-Fc. Means ± SEM of three independent experiments, n = 504 neurons. (D,E) DIV7 neurons bound with Caspr2-Fc (green) were double-stained for MAP2 (D, blue) or tau (E, red). The insets in (D’,E’) show that Caspr2-Fc preferentially bound on MAP2-positive dendrites and not on tau-positive axons. (A,B)z-stacks of six confocal sections with z-step of 0.5 μm. (D,E) Single optical sections of confocal images. Bar is in (A,B), 20 μm; in (D,E), 10 μm; in insets, 7 μm.
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Figure 6: The binding sites of Caspr2-Fc are localized on the somato-dendritic compartment. Hippocampal neurons at DIV4 (A–C) and DIV7 (D,E) were incubated with 10 μg/ml Caspr2-Fc preclustered with Alexa-conjugated anti-Fc IgGs for 30 min at 37°C. (A,B) DIV4 neurons bound with Caspr2-Fc (red). Cells were fixed and permeabilized before double-staining for MAP2 (blue) and GAD65 (green). (A,B) show representative images of GAD65-negative neurons (white arrow, A) and GAD65-positive neurons (green arrow, B) labeled with Caspr2-Fc. (C) Quantitative analysis of the percentage of total neurons, GAD65-positive and GAD65-negative neurons that were surface labeled for Caspr2-Fc. Means ± SEM of three independent experiments, n = 504 neurons. (D,E) DIV7 neurons bound with Caspr2-Fc (green) were double-stained for MAP2 (D, blue) or tau (E, red). The insets in (D’,E’) show that Caspr2-Fc preferentially bound on MAP2-positive dendrites and not on tau-positive axons. (A,B)z-stacks of six confocal sections with z-step of 0.5 μm. (D,E) Single optical sections of confocal images. Bar is in (A,B), 20 μm; in (D,E), 10 μm; in insets, 7 μm.
Mentions: Caspr2 belongs to the family of neurexins, which are pre-synaptic CAMs. Studies in culture indicate that neurexins are implicated in synaptogenesis by inducing the clustering of post-synaptic neuroligins (Dean et al., 2003; Craig and Kang, 2007). Neurexin/neuroligin association promotes the formation of excitatory and inhibitory synapses by interacting with PSD95 or Gephyrin, respectively. We asked whether Caspr2 might be also involved in trans-synaptic contacts. With this aim, we generated a Caspr2-Fc chimera to detect Caspr2 binding sites in hippocampal neuronal culture. Caspr2-Fc plasmid was transfected in HEK cells and the recombinant protein purified from the culture supernatant using Protein A-affinity chromatography. The chimera pre-clustered with fluorescent anti-Fc IgG was incubated with hippocampal neurons at DIV4 (Figure 6). We observed that Caspr2 binding sites were present on both GAD65-negative (arrow in Figure 6A) and GAD65-positive neurons (green arrow in Figure 6B). Quantitative analysis indicated that Caspr2-Fc bound 36% of the total neurons (Figure 6C). Caspr2-Fc bound to the somato-dendritic compartment of DIV7 neurons as determined using double-staining for MAP2 (Figures 6D,D’). In contrast, Caspr2-Fc was not co-localized with axons immunostained with anti-tau mAb (Figures 6E,E’). Caspr2-Fc binding sites were distributed on the somato-dendritic compartment of both inhibitory (26 ± 6%) and excitatory (38 ± 5%) neurons as analyzed at DIV4 (n = 504 neurons, three coverslips; Figure 6C).

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