Limits...
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

(A) Neurons were transfected with Gephyrin-GFP (Geph-GFP, green) at DIV14 and labeled at DIV21 for surface Caspr2 (red) and GAD65 (blue). (A’) The insets show the pre-synaptic sites double-labeled for Caspr2 and GAD65 facing post-synaptic clusters of Gephyrin-GFP (arrowheads). (B) Hippocampal neurons were transfected with Gephyrin-GFP (green) at DIV14 and incubated at DIV17 with control, LE5 or LE6 IgGs (1/100 dilution) for 1 h at 37°C. Clusters of post-synaptic Gephyrin in contact with presynaptic GAD65 are indicated with white arrows and non-synaptic Gephyrin with red arrows. (C) Quantitative analysis of the ratio of synaptic relative to total Gephyrin clusters under the different experimental conditions: incubation with culture medium (CTL), or with control, LE5 or LE6 IgGs. (D) Number of synaptic Gephyrin-GFP clusters per neuron. Means ± SEM, n indicates the number of neurons analyzed. ∗indicates significant difference (P < 0.05) with the culture medium condition using ANOVA followed by Fischer’s test. (E) Double-staining for surface Caspr2 (red) and VGAT as a marker of inhibitory pre-synaptic terminals with the box enlarged in (E’). Note the colocalisation of Caspr2 and VGAT indicated with arrowheads. (A,A’, B,E): Z-stack of five confocal sections with z-step of 0.5 μm. (E’) is a single confocal section. Bar is in (A,B,E), 10 μm; in insets (A’,E’), 1.5 μm.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4496579&req=5

Figure 5: (A) Neurons were transfected with Gephyrin-GFP (Geph-GFP, green) at DIV14 and labeled at DIV21 for surface Caspr2 (red) and GAD65 (blue). (A’) The insets show the pre-synaptic sites double-labeled for Caspr2 and GAD65 facing post-synaptic clusters of Gephyrin-GFP (arrowheads). (B) Hippocampal neurons were transfected with Gephyrin-GFP (green) at DIV14 and incubated at DIV17 with control, LE5 or LE6 IgGs (1/100 dilution) for 1 h at 37°C. Clusters of post-synaptic Gephyrin in contact with presynaptic GAD65 are indicated with white arrows and non-synaptic Gephyrin with red arrows. (C) Quantitative analysis of the ratio of synaptic relative to total Gephyrin clusters under the different experimental conditions: incubation with culture medium (CTL), or with control, LE5 or LE6 IgGs. (D) Number of synaptic Gephyrin-GFP clusters per neuron. Means ± SEM, n indicates the number of neurons analyzed. ∗indicates significant difference (P < 0.05) with the culture medium condition using ANOVA followed by Fischer’s test. (E) Double-staining for surface Caspr2 (red) and VGAT as a marker of inhibitory pre-synaptic terminals with the box enlarged in (E’). Note the colocalisation of Caspr2 and VGAT indicated with arrowheads. (A,A’, B,E): Z-stack of five confocal sections with z-step of 0.5 μm. (E’) is a single confocal section. Bar is in (A,B,E), 10 μm; in insets (A’,E’), 1.5 μm.

Mentions: Hippocampal neurons were transfected at DIV14 with Gephyrin-GFP to visualize the post-synaptic clusters facing pre-synaptic inhibitory contacts at DIV21 (Figure 5A). Gephyrin is the major post-synaptic scaffolding protein at inhibitory synapses (Craig et al., 1996). As shown in Figures 5A’, pre-synaptic terminals positive for both GAD65 and Caspr2 were apposed to Gephyrin-GFP clusters (arrowheads). In addition, surface Caspr2 colocalized with the inhibitory presynaptic terminals that were labeled for VGAT (Figures 5E,E’). We asked whether the LE patient’s autoantibodies directed against Caspr2 could display functional blocking activity by destabilizing inhibitory synaptic contacts. The LE5 and LE6 autoantibodies were tested which are mainly of the IgG4 isotype. LE5 IgGs are directed against multiple domains of Caspr2 and LE6 IgGs only target the N-terminal modules. Hippocampal neurons were transfected with Gephyrin-GFP at DIV14 and incubated at DIV17 with the culture medium, control IgGs, LE5, or LE6 IgGs diluted 1:100 for 1 h at 37°C. Using automatic spot detection of the Imaris software, we determined under each condition the number of total Gephyrin-GFP clusters and the number of Gephyrin-GFP clusters apposed to GAD65-positive presynaptic terminals (Figure 5B, white arrows). The ratio of synaptic versus total Gephyrin-GFP clusters was not significantly affected by incubation during 1 h with LE autoantibodies (Figure 5C). However, a significant decrease in the number of synaptic Gephyrin clusters per neuron was observed for LE5 and LE6 (24.5 and 30%, respectively, P < 0.05 using ANOVA and Fisher’s test) but not for control IgGs (10%) by comparison with culture medium incubation (Figure 5D). The number of GAD65-positive clusters contacting the somato-dendritic compartment per Gephyrin-GFP transfected neuron was not significantly decreased (284 ± 41 with culture medium, 230 ± 18 with control IgGs, 218 ± 21 with LE5, 210 ± 30 with LE6). Since Caspr2 was only expressed in 60% of GAD65-positive neurons, the functional effect of LE autoantibodies may be underestimated. In conclusion, we observed that Caspr2 is selectively localized along GABAergic axons and at the inhibitory pre-synaptic terminals in cultured hippocampal neurons. In addition, the perturbating assays of post-synaptic Gephyrin clusters suggest that anti-Caspr2 autoantibodies of LE patients may be pathogenic by altering the inhibitory synaptic contacts.


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)

(A) Neurons were transfected with Gephyrin-GFP (Geph-GFP, green) at DIV14 and labeled at DIV21 for surface Caspr2 (red) and GAD65 (blue). (A’) The insets show the pre-synaptic sites double-labeled for Caspr2 and GAD65 facing post-synaptic clusters of Gephyrin-GFP (arrowheads). (B) Hippocampal neurons were transfected with Gephyrin-GFP (green) at DIV14 and incubated at DIV17 with control, LE5 or LE6 IgGs (1/100 dilution) for 1 h at 37°C. Clusters of post-synaptic Gephyrin in contact with presynaptic GAD65 are indicated with white arrows and non-synaptic Gephyrin with red arrows. (C) Quantitative analysis of the ratio of synaptic relative to total Gephyrin clusters under the different experimental conditions: incubation with culture medium (CTL), or with control, LE5 or LE6 IgGs. (D) Number of synaptic Gephyrin-GFP clusters per neuron. Means ± SEM, n indicates the number of neurons analyzed. ∗indicates significant difference (P < 0.05) with the culture medium condition using ANOVA followed by Fischer’s test. (E) Double-staining for surface Caspr2 (red) and VGAT as a marker of inhibitory pre-synaptic terminals with the box enlarged in (E’). Note the colocalisation of Caspr2 and VGAT indicated with arrowheads. (A,A’, B,E): Z-stack of five confocal sections with z-step of 0.5 μm. (E’) is a single confocal section. Bar is in (A,B,E), 10 μm; in insets (A’,E’), 1.5 μm.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4496579&req=5

Figure 5: (A) Neurons were transfected with Gephyrin-GFP (Geph-GFP, green) at DIV14 and labeled at DIV21 for surface Caspr2 (red) and GAD65 (blue). (A’) The insets show the pre-synaptic sites double-labeled for Caspr2 and GAD65 facing post-synaptic clusters of Gephyrin-GFP (arrowheads). (B) Hippocampal neurons were transfected with Gephyrin-GFP (green) at DIV14 and incubated at DIV17 with control, LE5 or LE6 IgGs (1/100 dilution) for 1 h at 37°C. Clusters of post-synaptic Gephyrin in contact with presynaptic GAD65 are indicated with white arrows and non-synaptic Gephyrin with red arrows. (C) Quantitative analysis of the ratio of synaptic relative to total Gephyrin clusters under the different experimental conditions: incubation with culture medium (CTL), or with control, LE5 or LE6 IgGs. (D) Number of synaptic Gephyrin-GFP clusters per neuron. Means ± SEM, n indicates the number of neurons analyzed. ∗indicates significant difference (P < 0.05) with the culture medium condition using ANOVA followed by Fischer’s test. (E) Double-staining for surface Caspr2 (red) and VGAT as a marker of inhibitory pre-synaptic terminals with the box enlarged in (E’). Note the colocalisation of Caspr2 and VGAT indicated with arrowheads. (A,A’, B,E): Z-stack of five confocal sections with z-step of 0.5 μm. (E’) is a single confocal section. Bar is in (A,B,E), 10 μm; in insets (A’,E’), 1.5 μm.
Mentions: Hippocampal neurons were transfected at DIV14 with Gephyrin-GFP to visualize the post-synaptic clusters facing pre-synaptic inhibitory contacts at DIV21 (Figure 5A). Gephyrin is the major post-synaptic scaffolding protein at inhibitory synapses (Craig et al., 1996). As shown in Figures 5A’, pre-synaptic terminals positive for both GAD65 and Caspr2 were apposed to Gephyrin-GFP clusters (arrowheads). In addition, surface Caspr2 colocalized with the inhibitory presynaptic terminals that were labeled for VGAT (Figures 5E,E’). We asked whether the LE patient’s autoantibodies directed against Caspr2 could display functional blocking activity by destabilizing inhibitory synaptic contacts. The LE5 and LE6 autoantibodies were tested which are mainly of the IgG4 isotype. LE5 IgGs are directed against multiple domains of Caspr2 and LE6 IgGs only target the N-terminal modules. Hippocampal neurons were transfected with Gephyrin-GFP at DIV14 and incubated at DIV17 with the culture medium, control IgGs, LE5, or LE6 IgGs diluted 1:100 for 1 h at 37°C. Using automatic spot detection of the Imaris software, we determined under each condition the number of total Gephyrin-GFP clusters and the number of Gephyrin-GFP clusters apposed to GAD65-positive presynaptic terminals (Figure 5B, white arrows). The ratio of synaptic versus total Gephyrin-GFP clusters was not significantly affected by incubation during 1 h with LE autoantibodies (Figure 5C). However, a significant decrease in the number of synaptic Gephyrin clusters per neuron was observed for LE5 and LE6 (24.5 and 30%, respectively, P < 0.05 using ANOVA and Fisher’s test) but not for control IgGs (10%) by comparison with culture medium incubation (Figure 5D). The number of GAD65-positive clusters contacting the somato-dendritic compartment per Gephyrin-GFP transfected neuron was not significantly decreased (284 ± 41 with culture medium, 230 ± 18 with control IgGs, 218 ± 21 with LE5, 210 ± 30 with LE6). Since Caspr2 was only expressed in 60% of GAD65-positive neurons, the functional effect of LE autoantibodies may be underestimated. In conclusion, we observed that Caspr2 is selectively localized along GABAergic axons and at the inhibitory pre-synaptic terminals in cultured hippocampal neurons. In addition, the perturbating assays of post-synaptic Gephyrin clusters suggest that anti-Caspr2 autoantibodies of LE patients may be pathogenic by altering the inhibitory synaptic contacts.

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