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

Distribution of Caspr2 at pre-synaptic sites of excitatory and inhibitory axons. Confocal images of hippocampal neurons at DIV14 (A,B) or DIV21 (C) that were surface labeled for Caspr2 using LE1 IgGs (red). Cells were fixed and permeabilized before double-staining for MAP2 (blue), vGLUT1 (A, green) or GAD65 (B,C, green). (A’) Enlarged areas shows glutamatergic pre-synaptic sites stained for Caspr2. (B’,C’–C”’) Inhibitory pre-synaptic sites labeled for GAD65 (arrowheads) were intensely stained for Caspr2 at the contact with the soma (C”) or dendrites (B’,C”’). Note that GAD65-positive axons surrounding the soma of pyramidal neurons were heavily stained for Caspr2 (yellow arrows). (C’) This enlarged area shows Caspr2 punctate immunostaining along a GAD65-positive axon apposed to a dendrite. Bar is in (A–C), 9 μm; in insets, 1.5 μm.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 4: Distribution of Caspr2 at pre-synaptic sites of excitatory and inhibitory axons. Confocal images of hippocampal neurons at DIV14 (A,B) or DIV21 (C) that were surface labeled for Caspr2 using LE1 IgGs (red). Cells were fixed and permeabilized before double-staining for MAP2 (blue), vGLUT1 (A, green) or GAD65 (B,C, green). (A’) Enlarged areas shows glutamatergic pre-synaptic sites stained for Caspr2. (B’,C’–C”’) Inhibitory pre-synaptic sites labeled for GAD65 (arrowheads) were intensely stained for Caspr2 at the contact with the soma (C”) or dendrites (B’,C”’). Note that GAD65-positive axons surrounding the soma of pyramidal neurons were heavily stained for Caspr2 (yellow arrows). (C’) This enlarged area shows Caspr2 punctate immunostaining along a GAD65-positive axon apposed to a dendrite. Bar is in (A–C), 9 μm; in insets, 1.5 μm.

Mentions: We also examined how Caspr2 was distributed along axons and pre-synaptic sites at DIV14 and DIV21 later stages. The vesicular glutamate transporter-1 vGLUT1 was used as a marker for glutamatergic axons and synapses. As shown in Figure 4A, some of the vGLUT1-positive axons at DIV14 were labeled for Caspr2 using LE1 serum IgGs. High magnification images show that Caspr2 co-localized with vGLUT1 at pre-synaptic sites (Figures 4A’, arrowheads). Next we used GAD65, which synthesizes GABA for neurotransmission as a marker for inhibitory axons and synapses. Caspr2 strongly co-localized with GAD65-positive axons as observed using serum IgGs of all the patients analyzed (Supplementary Figure S2). In particular, the GAD65-positive axons were surrounding the MAP2-labeled large pyramidal neurons and were heavily stained for Caspr2 at DIV14 and DIV21 as shown using LE1 serum IgGs (arrows in Figures 4B,C). The presynaptic sites labeled for GAD65 were intensely stained for Caspr2 at the contact with the soma (arrowheads in Figures 4C”) or dendrites (arrowheads in Figures 4B’,C”’). We estimated that 51% of the GABAergic pre-synaptic contacts on dendrites were labeled for Caspr2 (5.9 ± 0.5 Caspr2-positive of 11.6 ± 0.5 total GABAergic contacts/25 μm dendritic length; n = 14 dendrites). As illustrated in Figures 4C’, Caspr2 was distributed along inhibitory axons partially overlapping with GAD65 puncta. We estimated that 34 % of the Caspr2-positive clusters contacting dendrites were GAD65-positive (13.7 ± 2 Caspr2-positive clusters among which 4.7 ± 0.8 were GABAergic/25 μm).


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)

Distribution of Caspr2 at pre-synaptic sites of excitatory and inhibitory axons. Confocal images of hippocampal neurons at DIV14 (A,B) or DIV21 (C) that were surface labeled for Caspr2 using LE1 IgGs (red). Cells were fixed and permeabilized before double-staining for MAP2 (blue), vGLUT1 (A, green) or GAD65 (B,C, green). (A’) Enlarged areas shows glutamatergic pre-synaptic sites stained for Caspr2. (B’,C’–C”’) Inhibitory pre-synaptic sites labeled for GAD65 (arrowheads) were intensely stained for Caspr2 at the contact with the soma (C”) or dendrites (B’,C”’). Note that GAD65-positive axons surrounding the soma of pyramidal neurons were heavily stained for Caspr2 (yellow arrows). (C’) This enlarged area shows Caspr2 punctate immunostaining along a GAD65-positive axon apposed to a dendrite. Bar is in (A–C), 9 μm; in insets, 1.5 μm.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 4: Distribution of Caspr2 at pre-synaptic sites of excitatory and inhibitory axons. Confocal images of hippocampal neurons at DIV14 (A,B) or DIV21 (C) that were surface labeled for Caspr2 using LE1 IgGs (red). Cells were fixed and permeabilized before double-staining for MAP2 (blue), vGLUT1 (A, green) or GAD65 (B,C, green). (A’) Enlarged areas shows glutamatergic pre-synaptic sites stained for Caspr2. (B’,C’–C”’) Inhibitory pre-synaptic sites labeled for GAD65 (arrowheads) were intensely stained for Caspr2 at the contact with the soma (C”) or dendrites (B’,C”’). Note that GAD65-positive axons surrounding the soma of pyramidal neurons were heavily stained for Caspr2 (yellow arrows). (C’) This enlarged area shows Caspr2 punctate immunostaining along a GAD65-positive axon apposed to a dendrite. Bar is in (A–C), 9 μm; in insets, 1.5 μm.
Mentions: We also examined how Caspr2 was distributed along axons and pre-synaptic sites at DIV14 and DIV21 later stages. The vesicular glutamate transporter-1 vGLUT1 was used as a marker for glutamatergic axons and synapses. As shown in Figure 4A, some of the vGLUT1-positive axons at DIV14 were labeled for Caspr2 using LE1 serum IgGs. High magnification images show that Caspr2 co-localized with vGLUT1 at pre-synaptic sites (Figures 4A’, arrowheads). Next we used GAD65, which synthesizes GABA for neurotransmission as a marker for inhibitory axons and synapses. Caspr2 strongly co-localized with GAD65-positive axons as observed using serum IgGs of all the patients analyzed (Supplementary Figure S2). In particular, the GAD65-positive axons were surrounding the MAP2-labeled large pyramidal neurons and were heavily stained for Caspr2 at DIV14 and DIV21 as shown using LE1 serum IgGs (arrows in Figures 4B,C). The presynaptic sites labeled for GAD65 were intensely stained for Caspr2 at the contact with the soma (arrowheads in Figures 4C”) or dendrites (arrowheads in Figures 4B’,C”’). We estimated that 51% of the GABAergic pre-synaptic contacts on dendrites were labeled for Caspr2 (5.9 ± 0.5 Caspr2-positive of 11.6 ± 0.5 total GABAergic contacts/25 μm dendritic length; n = 14 dendrites). As illustrated in Figures 4C’, Caspr2 was distributed along inhibitory axons partially overlapping with GAD65 puncta. We estimated that 34 % of the Caspr2-positive clusters contacting dendrites were GAD65-positive (13.7 ± 2 Caspr2-positive clusters among which 4.7 ± 0.8 were GABAergic/25 μm).

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