Selective Axonal Expression of the Kv1 Channel Complex in Pre-myelinated GABAergic Hippocampal Neurons.

In myelinated fibers, the voltage-gated sodium channels Nav1 are concentrated at the nodal gap to ensure the saltatory propagation of action potentials. The voltage-gated potassium channels Kv1 are segregated at the juxtaparanodes under the compact myelin sheath and may stabilize axonal conduction. It has been recently reported that hippocampal GABAergic neurons display high density of Nav1 channels remarkably in clusters along the axon before myelination (Freeman et al., 2015). In inhibitory neurons, the Nav1 channels are trapped by the ankyrinG scaffold at the axon initial segment (AIS) as observed in pyramidal and granule neurons, but are also forming "pre-nodes," which may accelerate conduction velocity in pre-myelinated axons. However, the distribution of the Kv1 channels along the pre-myelinated inhibitory axons is still unknown. In the present study, we show that two subtypes of hippocampal GABAergic neurons, namely the somatostatin and parvalbumin positive cells, display a selective high expression of Kv1 channels at the AIS and all along the unmyelinated axons. These inhibitory axons are also highly enriched in molecules belonging to the juxtaparanodal Kv1 complex, including the cell adhesion molecules (CAMs) TAG-1, Caspr2, and ADAM22 and the scaffolding protein 4.1B. Here, taking advantage of hippocampal cultures from 4.1B and TAG-1 knock-out mice, we observed that 4.1B is required for the proper positioning of Caspr2 and TAG-1 along the distal axon, and that TAG-1 deficiency induces alterations in the axonal distribution of Caspr2. However, the axonal expression of Kv1 channels and clustering of ankyrinG were not modified. In conclusion, this study allowed the analysis of the hierarchy between channels, CAMs and scaffolding proteins for their expression along hippocampal inhibitory axons before myelination. The early steps of channel compartmentalization preceding myelination may be crucial for stabilizing nerve impulses switching from a continuous to saltatory conduction during network development.

ADAM22 and ADAM23 modulate the targeting of the Kv1 channel-associated protein LGI1 to the axon initial segment.

The distribution of the voltage-gated Kv1 K+ channels at the axon initial segment (AIS) influences neuronal intrinsic excitability. The Kv1.1 and Kv1.2 (also known as KCNA1 and KCNA2, respectively) subunits are associated with cell adhesion molecules (CAMs), including Caspr2 (also known as CNTNAP2) and LGI1, which are implicated in autoimmune and genetic neurological diseases with seizures. In particular, mutations in the LGI1 gene cause autosomal dominant lateral temporal lobe epilepsy (ADLTE). Here, by using rat hippocampal neurons in culture, we showed that LGI1 is recruited to the AIS where it colocalizes with ADAM22 and Kv1 channels. Strikingly, the missense mutations S473L and R474Q of LGI1 identified in ADLTE prevent its association with ADAM22 and enrichment at the AIS. Moreover, we observed that ADAM22 and ADAM23 modulate the trafficking of LGI1, and promote its ER export and expression at the overall neuronal cell surface. Live-cell imaging indicated that LGI1 is co-transported in axonal vesicles with ADAM22 and ADAM23. Finally, we showed that ADAM22 and ADAM23 also associate with Caspr2 and TAG-1 (also known as CNTN2) to be selectively targeted to different axonal sub-regions. Hence, the combinatorial expression of Kv1-associated CAMs may be critical to tune intrinsic excitability in physiological and epileptogenic contexts.

Genetic variants in autism-related CNTNAP2 impair axonal growth of cortical neurons.

The CNTNAP2 gene, coding for the cell adhesion glycoprotein Caspr2, is thought to be one of the major susceptibility genes for autism spectrum disorder (ASD). A large number of rare heterozygous missense CNTNAP2 variants have been identified in ASD patients. However, most of them are inherited from an unaffected parent, questioning their clinical significance. In the present study, we evaluate their impact on neurodevelopmental functions of Caspr2 in a heterozygous genetic background. Performing cortical neuron cultures from mouse embryos, we demonstrate that Caspr2 plays a dose-dependent role in axon growth in vitro. Loss of one Cntnap2 allele is sufficient to elicit axonal growth alteration, revealing a situation that may be relevant for CNTNAP2 heterozygosity in ASD patients. Then, we show that the two ASD variants I869T and G731S, which present impaired binding to Contactin2/TAG-1, do not rescue axonal growth deficits. We find that the variant R1119H leading to protein trafficking defects and retention in the endoplasmic reticulum has a dominant-negative effect on heterozygous Cntnap2 cortical neuron axon growth, through oligomerization with wild-type Caspr2. Finally, we identify an additional variant (N407S) with a dominant-negative effect on axon growth although it is well-localized at the membrane and properly binds to Contactin2. Thus, our data identify a new neurodevelopmental function for Caspr2, the dysregulation of which may contribute to clinical manifestations of ASD, and provide evidence that CNTNAP2 heterozygous missense variants may contribute to pathogenicity in ASD, through selective mechanisms.

The Kv1-associated molecules TAG-1 and Caspr2 are selectively targeted to the axon initial segment in hippocampal neurons.

Caspr2 and TAG-1 (also known as CNTNAP2 and CNTN2, respectively) are cell adhesion molecules (CAMs) associated with the voltage-gated potassium channels Kv1.1 and Kv1.2 (also known as KCNA1 and KCNA2, respectively) at regions controlling axonal excitability, namely, the axon initial segment (AIS) and juxtaparanodes of myelinated axons. The distribution of Kv1 at juxtaparanodes requires axo-glial contacts mediated by Caspr2 and TAG-1. In the present study, we found that TAG-1 strongly colocalizes with Kv1.2 at the AIS of cultured hippocampal neurons, whereas Caspr2 is uniformly expressed along the axolemma. Live-cell imaging revealed that Caspr2 and TAG-1 are sorted together in axonal transport vesicles. Therefore, their differential distribution may result from diffusion and trapping mechanisms induced by selective partnerships. By using deletion constructs, we identified two molecular determinants of Caspr2 that regulate its axonal positioning. First, the LNG2-EGF1 modules in the ectodomain of Caspr2, which are involved in its axonal distribution. Deletion of these modules promotes AIS localization and association with TAG-1. Second, the cytoplasmic PDZ-binding site of Caspr2, which could elicit AIS enrichment and recruitment of the membrane-associated guanylate kinase (MAGuK) protein MPP2. Hence, the selective distribution of Caspr2 and TAG-1 may be regulated, allowing them to modulate the strategic function of the Kv1 complex along axons.

Assembly of juxtaparanodes in myelinating DRG culture: Differential clustering of the Kv1/Caspr2 complex and scaffolding protein 4.1B.

The precise distribution of ion channels at the nodes of Ranvier is essential for the efficient propagation of action potentials along myelinated axons. The voltage-gated potassium channels Kv1.1/1.2 are clustered at the juxtaparanodes in association with the cell adhesion molecules, Caspr2 and TAG-1 and the scaffolding protein 4.1B. In the present study, we set up myelinating cultures of DRG neurons and Schwann cells to look through the formation of juxtaparanodes in vitro. We showed that the Kv1.1/Kv1.2 channels were first enriched at paranodes before being restricted to distal paranodes and juxtaparanodes. In addition, the Kv1 channels displayed an asymmetric expression enriched at the distal juxtaparanodes. Caspr2 was strongly co-localized with Kv1.2 whereas the scaffolding protein 4.1B was preferentially recruited at paranodes while being present at juxtaparanodes too. Kv1.2/Caspr2 but not 4.1B, also transiently accumulated within the nodal region both in myelinated cultures and developing sciatic nerves. Studying cultures and sciatic nerves from 4.1B KO mice, we further showed that 4.1B is required for the proper targeting of Caspr2 early during myelination. Moreover, using adenoviral-mediated expression of Caspr-GFP and photobleaching experiments, we analyzed the stability of paranodal junctions and showed that the lateral stability of paranodal Caspr was not altered in 4.1B KO mice indicating that 4.1B is not required for the assembly and stability of the paranodal junctions. Thus, developing an adapted culture paradigm, we provide new insights into the dynamic and differential distribution of Kv1 channels and associated proteins during myelination.

Inhibitory axons are targeted in hippocampal cell culture by anti-Caspr2 autoantibodies associated with limbic encephalitis.

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.

Specific contactin N-glycans are implicated in neurofascin binding and autoimmune targeting in peripheral neuropathies.

Cell adhesion molecules (CAMs) play a crucial role in the formation of the nodes of Ranvier and in the rapid propagation of the nerve impulses along myelinated axons. These CAMs are the targets of autoimmunity in inflammatory neuropathies. We recently showed that a subgroup of patients with aggressive chronic inflammatory demyelinating polyradiculoneuropathy (CIDP) shows autoantibodies to contactin (1). The complex of contactin·Caspr·neurofascin-155 (NF155) enables the formation of paranodal junctions, suggesting that antibody attack against paranodes may participate in the severity of CIDP. In the present study, we mapped the molecular determinants of contactin targeted by the autoantibodies. In three patients, immunoreactivity was directed against the Ig domains of contactin and was dependent on N-glycans. The serum of one patient was selectively directed against contactin bearing mannose-rich N-glycans. Strikingly, the oligomannose type sugars of contactin are required for association with its glial partner NF155 (2). To investigate precisely the role of contactin N-glycans, we have mutated each of the nine consensus N-glycosylation sites independently. We found that the mutation of three sites (N467Q/N473Q/N494Q) in Ig domain 5 of contactin prevented soluble NF155-Fc binding. In contrast, these mutations did not abolish cis-association with Caspr. Next, we showed that the cluster of N-glycosylation sites (Asn-467, Asn-473, and Asn-494) was required for immunoreactivity in one patient. Using cell aggregation assays, we showed that the IgGs from the four CIDP patients prevented adhesive interaction between contactin·Caspr and NF155. Importantly, we showed that the anti-contactin autoantibodies induced alteration of paranodal junctions in myelinated neuronal culture. These results strongly suggest that antibodies to CAMs may be pathogenic and induce demyelination via functional blocking activity.

Altered heart rate control in transgenic mice carrying the KCNJ6 gene of the human chromosome 21.

Congenital heart defects (CHD) are common in Down syndrome (DS, trisomy 21). Recently, cardiac sympathetic-parasympathetic imbalance has also been documented in DS adults free of any CHD. The KCNJ6 gene located on human chromosome 21 encodes for the Kir3.2/GIRK2 protein subunits of G protein-regulated K(+) (K(G)) channels and could contribute to this altered cardiac regulation. To elucidate the role of its overexpression, we used homozygous transgenic (Tg(+/+)) mice carrying copies of human KCNJ6. These mice showed human Kir3.2 mRNA expression in the heart and a 2.5-fold increased translation in the atria. Phenotypic alterations were assessed by recording electrocardiogram of urethane anesthetized mice. Chronotropic responses to direct (carbachol) and indirect (methoxamine) muscarinic stimulation were enhanced in Tg(+/+) mice with respect to wild-type (WT) mice. Alternating periods of slow and fast rhythm induced by CCPA (2-chloro-N-cyclopentyl-adenosine) were amplified in Tg(+/+) mice, resulting in a reduced negative chronotropic effect. These drugs reduced the atrial P wave amplitude and area. P wave variations induced by methoxamine and CCPA were respectively increased and reduced in the Tg(+/+) mice, while PR interval and ventricular wave showed no difference between Tg(+/+) and WT. These results indicate that Tg(+/+) mice incorporating the human KCNJ6 exhibit altered Kir3.2 expression and responses to drugs that would activate K(G) channels. Moreover, these altered expression and responses are limited to sino-atrial node and atria that normally express large amounts of K(G) channels. These data suggest that KCNJ6 could play an important role in altered cardiac regulation in DS patients.

Modal gating of human CaV2.1 (P/Q-type) calcium channels: I. The slow and the fast gating modes and their modulation by beta subunits.

The single channel gating properties of human CaV2.1 (P/Q-type) calcium channels and their modulation by the auxiliary beta1b, beta2e, beta3a, and beta4a subunits were investigated with cell-attached patch-clamp recordings on HEK293 cells stably expressing human CaV2.1 channels. These calcium channels showed a complex modal gating, which is described in this and the following paper (Fellin, T., S. Luvisetto, M. Spagnolo, and D. Pietrobon. 2004. J. Gen. Physiol. 124:463-474). Here, we report the characterization of two modes of gating of human CaV2.1 channels, the slow mode and the fast mode. A channel in the two gating modes differs in mean closed times and latency to first opening (both longer in the slow mode), in voltage dependence of the open probability (larger depolarizations are necessary to open the channel in the slow mode), in kinetics of inactivation (slower in the slow mode), and voltage dependence of steady-state inactivation (occurring at less negative voltages in the slow mode). CaV2.1 channels containing any of the four beta subtypes can gate in either the slow or the fast mode, with only minor differences in the rate constants of the transitions between closed and open states within each mode. In both modes, CaV2.1 channels display different rates of inactivation and different steady-state inactivation depending on the beta subtype. The type of beta subunit also modulates the relative occurrence of the slow and the fast gating mode of CaV2.1 channels; beta3a promotes the fast mode, whereas beta4a promotes the slow mode. The prevailing mode of gating of CaV2.1 channels lacking a beta subunit is a gating mode in which the channel shows shorter mean open times, longer mean closed times, longer first latency, a much larger fraction of nulls, and activates at more positive voltages than in either the fast or slow mode.

Robo1 and Robo2 are homophilic binding molecules that promote axonal growth.

In the present study we show that following transfection in 3T3 cells, human Robo1 and Robo2 stimulate neurite outgrowth from Robo-positive neurons (retinal neurons and olfactory neurons), but have no effect on Robo-negative neurons (cerebellar granule cells). The neurite outgrowth response was inhibited by an antibody raised against the first Ig domain of Robo1/2 or by soluble Robo-Fc chimera. Furthermore, we show that the extracellular domains of Robo1 and Robo2 are homophilic adhesion molecules that can also interact with each other. These data suggest a wider range of functions for the Robo family in the development of the nervous system and provide novel insights into the molecular basis for the phenotypes observed in Robo mutants in Drosophila, C. elegans, and zebrafish.