Effect of monovalency on anti-contactin-1 IgG4.

Introduction: Autoimmune nodopathies (AN) have been diagnosed in a subset of patients fulfilling criteria for chronic inflammatory demyelinating polyradiculoneuropathy (CIDP) who display no or poor response to intravenous immunoglobulins. Biomarkers of AN are autoantibodies, mainly IgG4, directed against the ternary paranodal complex composed by neurofascin-155, contactin-1 (CNTN1), and Contactin-associated-protein-1 (CASPR1) or against the nodal isoforms of neurofascin. IgG4 can undergo a Fab-arm exchange (FAE) which results in functionally monovalent antibody. This phenomenon differentially affects the pathogenicity of IgG4 depending on the target of autoantibodies. Here, we have evaluated this issue by examining the impact of valency on anti-CNTN1 IgG4 which induces paranodal destruction through a function blocking activity. Methods: Sera were obtained from 20 patients with AN associated with anti-CNTN1 antibodies. The proportion of monospecific/bispecific anti-CNTN1 antibodies was estimated in each patient by ELISA by examining the ability of serum antibodies to cross-link untagged CNTN1 with biotinylated CNTN1. To determine the impact of monovalency, anti-CNTN1 IgG4 were enzymatically digested into monovalent Fab and tested in vitro on cell aggregation assay. Also, intraneural injections were performed to determine whether monovalent Fab and native IgG4 may penetrate paranode, and antibody infiltration was monitored 1- and 3-days post injection. Results and discussion: We found that the percentage of monospecific antibodies were lower than 5% in 14 out of 20 patients (70%), suggesting that IgG4 have undergone extensive FAE in situ. The levels of monospecific antibodies correlated with the titers of anti-CNTN1 antibodies. However, no correlation was found with clinical severity, and patients with low or high percentage of monospecific antibodies similarly showed a severe phenotype. Native anti-CNTN1 IgG4 were shown to inhibit the interaction between cells expressing CNTN1/CASPR1 and cells expressing neurofascin-155 using an in vitro aggregation assay. Similarly, monovalent Fab significantly inhibited the interaction between CNTN1/CASPR1 and neurofascin-155. Intraneural injections of Fab and native anti-CNTN1 IgG4 indicated that both mono- and bivalent anti-CNTN1 IgG4 potently penetrated the paranodal regions and completely invaded this region by day 3. Altogether, these data indicate anti-CNTN1 IgG4 are mostly bispecific in patients, and that functionally monovalent anti-CNTN1 antibodies have the pathogenic potency to alter paranode.

Immune-mediated diseases involving central and peripheral nervous systems.

BACKGROUND AND PURPOSE: In addition to combined central and peripheral demyelination, other immune diseases could involve both the central nervous system (CNS) and peripheral nervous system (PNS). METHODS: To identify immune-mediated diseases responsible for symptomatic combined central/peripheral nervous system involvement (ICCPs), we conducted a multicentric retrospective study and assessed clinical, electrophysiological, and radiological features of patients fulfilling our ICCP criteria. RESULTS: Thirty patients (20 males) were included and followed during a median of 79.5 months (interquartile range [IQR] = 43-145). The median age at onset was 51.5 years (IQR = 39-58). Patients were assigned to one of four groups: (i) monophasic disease with concomitant CNS/PNS involvement including anti-GQ1b syndrome (acute polyradiculoneuropathy + rhombencephalitis, n = 2), checkpoint inhibitor-related toxicities (acute polyradiculoneuropathy + encephalitis, n = 3), and anti-glial fibrillary acidic protein astrocytopathy (subacute polyradiculoneuropathy and meningoencephalomyelitis with linear gadolinium enhancements, n = 2); (ii) chronic course with concomitant CNS/PNS involvement including paraneoplastic syndromes (ganglionopathy/peripheral hyperexcitability + limbic encephalitis, n = 4); (iii) chronic course with sequential CNS/PNS involvement including POEMS (polyneuropathy, organomegaly, endocrinopathy, monoclonal protein, skin changes) syndrome (polyradiculoneuropathy + strokes, n = 2), histiocytosis (polyradiculoneuropathy + lepto-/pachymeningitis, n = 1), and systemic vasculitis (multineuropathy + CNS vasculitis/pachymeningitis, n = 2); and (iv) chronic course with concomitant or sequential CNS/PNS involvement including combined central and peripheral demyelination (polyradiculoneuropathy + CNS demyelinating lesions, n = 10) and connective tissue diseases (ganglionopathy/radiculopathy/multineuropathy + limbic encephalitis/transverse myelitis/stroke, n = 4). CONCLUSIONS: We diagnosed nine ICCPs. The timing of central and peripheral manifestations and the disease course help determine the underlying immune disease. When antibody against neuroglial antigen is identified, CNS and PNS involvement is systematically concomitant, suggesting a common CNS/PNS antigen and a simultaneous disruption of blood-nerve and blood-brain barriers.

Chronic inflammatory demyelinating polyneuropathy with anti-NF155 IgG4 in China.

Patients with chronic inflammatory demyelinating polyneuropathy (CIDP) seropositive for autoantibodies against nodal and paranodal proteins display distinct clinical presentations. We herein tested for autoantibodies against neurofascin (NF) 155, NF186, contactin-associated protein 1 and contactin-1 and investigated the autoantibody-related clinical features in 29 patients with CIDP from China. Six patients with anti-NF155 IgG4 antibodies displayed younger age of onset and poor response to intravenous immunoglobulin than seronegative patients. One patient had anti-NF186 IgG antibody and no patients had anti-contactin-associated protein 1 or anti-contactin-1 antibodies. Clinical features of CIDP patients with anti-NF155 antibodies in China were similar to those reported in other countries.

Anti-Neurofascin-155 IgG4 antibodies prevent paranodal complex formation in vivo.

Neurofascin-155 (Nfasc155) is an essential glial cell adhesion molecule expressed in paranodal septate-like junctions of peripheral and central myelinated axons. The genetic deletion of Nfasc155 results in the loss of septate-like junctions and in conduction slowing. In humans, IgG4 antibodies against Nfasc155 are implicated in the pathogenesis of chronic inflammatory demyelinating polyneuropathy (CIDP). These antibodies are associated with an aggressive onset, a refractoriness to intravenous immunoglobulin, and tremor of possible cerebellar origin. Here, we examined the pathogenic effects of patient-derived anti-Nfasc155 IgG4. These antibodies did not inhibit the ability of Nfasc155 to complex with its axonal partners contactin-1/CASPR1 or induce target internalization. Passive transfer experiments revealed that IgG4 antibodies target Nfasc155 on Schwann cell surface, and diminished Nfasc155 protein levels and prevented paranodal complex formation in neonatal animals. In adult animals, chronic intrathecal infusions of antibodies also induced the loss of Nfasc155 and of paranodal specialization and resulted in conduction alterations in motor nerves. These results indicate that anti-Nfasc155 IgG4 perturb conduction in absence of demyelination, validating the existence of paranodopathy. These results also shed light on the mechanisms regulating protein insertion at paranodes.

Acute painful autoimmune neuropathy: A variant of Guillain-Barré syndrome.

INTRODUCTION: We present a painful small-fiber neuropathy variant of Guillain-Barré syndrome characterized by antecedent infectious symptoms, hyporeflexia, and albuminocytologic dissociation. METHODS: Two patients received intravenous immunoglobulin, one corticosteroids. RESULTS: The patients subsequently improved. Immunoglobulin G (IgG) antibodies in their acute phase sera strongly bound to murine small nerve fibers, and the binding disappeared during the convalescent phase. Serum transfer to a murine nociceptive model induced transient alteration in thermal pain responses. DISCUSSION: Our case series suggest that an acute transient immune response can be directed against small nerve fibers, and that patients so affected can exhibit features of Guillain-Barré syndrome. Muscle Nerve 57: 320-324, 2018.

Autoantibodies to nodal isoforms of neurofascin in chronic inflammatory demyelinating polyneuropathy.

Chronic inflammatory demyelination polyneuropathy is a heterogeneous and treatable immune-mediated disorder that lacks biomarkers to support diagnosis. Recent evidence indicates that paranodal proteins (contactin 1, contactin-associated protein 1, and neurofascin-155) are the targets of autoantibodies in subsets of patients showing distinct clinical presentations. Here, we identified neurofascin-186 and neurofascin-140 as the main targets of autoantibodies in five patients presenting IgG reactivity against the nodes of Ranvier. Four patients displayed predominantly IgG4 antibodies, and one patient presented IgG3 antibodies that activated the complement pathway in vitro. These patients present distinct clinical features compared to those with anti-neurofascin-155 IgG4. Most patients had a severe phenotype associated with conduction block or decreased distal motor amplitude. Four patients had a subacute-onset and sensory ataxia. Two patients presented with nephrotic syndromes and one patient with an IgG4-related retroperitoneal fibrosis. Intravenous immunoglobulin and corticosteroids were effective in three patients, and one patient remitted following rituximab treatment. Clinical remission was associated with autoantibody depletion and with recovery of conduction block and distal motor amplitude suggesting a nodo-paranodopathy. Our data demonstrate that the pathogenic mechanisms responsible for chronic inflammatory demyelination polyneuropathy are broad and may include dysfunctions at the nodes of Ranvier in a subgroup of patients.

Paranodal lesions in chronic inflammatory demyelinating polyneuropathy associated with anti-Neurofascin 155 antibodies.

Antibodies to Contactin-1 and Neurofascin 155 (Nfasc155) have recently been associated with subsets of patients with chronic inflammatory demyelinating polyneuropathy (CIDP). Contactin-1 and Nfasc155 are cell adhesion molecules that constitute the septate-like junctions observed by electron microscopy in the paranodes of myelinated axons. Antibodies to Contactin-1 have been shown to affect the localization of paranodal proteins both in patient nerve biopsies and in animal models after passive transfer. However, it is unclear whether these antibodies alter the paranodal ultrastructure. We examined by electron microscopy sural nerve biopsies from two patients presenting with anti-Nfasc155 antibodies, and also four patients lacking antibodies, three normal controls, and five patients with other neuropathies. We found that patients with anti-Nfasc155 antibodies presented a selective loss of the septate-like junctions at all paranodes examined. Further, cellular processes penetrated into the expanded spaces between the paranodal myelin loops and the axolemma in these patients. These patients presented with important nerve conduction slowing and demyelination. Also, the reactivity of anti-Nfasc155 antibodies from these patients was abolished in neurofascin-deficient mice, confirming that the antibodies specifically target paranodal proteins. Our data indicate that anti-Nfasc155 destabilizes the paranodal axo-glial junctions and may participate in conduction deterioration.

Contactin-1 IgG4 antibodies cause paranode dismantling and conduction defects.

Paranodal axoglial junctions formed by the association of contactin-1, contactin-associated protein 1, and neurofascin-155, play important functions in nerve impulse propagation along myelinated axons. Autoantibodies to contactin-1 and neurofascin-155 define chronic inflammatory demyelinating polyradiculoneuropathy subsets of patients with specific clinical features. These autoantibodies are mostly of the IgG4 isotype, but their pathogenicity has not been proven. Here, we investigated the mechanisms how IgG subclasses to contactin-1 affect conduction. We show that purified anti-contactin-1 IgG1 and IgG4 bind to paranodes. To determine whether these isotypes can pass the paranodal barrier, we incubated isolated sciatic nerves with the purified antibody or performed intraneural injections. We found that IgG4 diffused into the paranodal regions in vitro or after intraneural injections. IgG4 infiltration was slow and progressive. In 24 h, IgG4 accessed the paranode borders near the nodal lumen, and completely fill the paranodal segments by 3 days. By contrast, control IgG, anti-contactin-1 IgG1, or even anti-contactin-associated-protein-2 IgG4 did not pass the paranodal barrier. To determine whether chronic exposure to these antibodies is pathogenic, we passively transferred anti-contactin-1 IgG1 and IgG4 into Lewis rats immunized with P2 peptide. IgG4 to contactin-1, but not IgG1, induced progressive clinical deteriorations combined with gait ataxia. No demyelination, axonal degeneration, or immune infiltration were observed. Instead, these animals presented a selective loss of the paranodal specialization in motor neurons characterized by the disappearance of the contactin-associated protein 1/contactin-1/neurofascin-155 complex at paranodes. Paranode destruction did not affect nodal specialization, but resulted in a moderate node lengthening. The sensory nerves and dorsal root ganglion were not affected in these animals. Electrophysiological examination further supported these results and revealed strong nerve activity loss affecting predominantly small diameter or slow conducting motor axons. These deficits partly matched with those found in patients: proximal motor involvement, gait ataxia, and a demyelinating neuropathy that showed early axonal features. The animal model thus seemed to replicate the early deteriorations in these patients and pointed out that paranodal loss in mature fibres results in conduction defects, but not conduction slowing. Our findings indicate that IgG4 directed against contactin-1 are pathogenic and are reliable biomarkers of a specific subset of chronic inflammatory demyelinating polyneuropathy patients. These antibodies appear to loosen the paranodal barrier, thereby favouring antibody progression and causing paranodal collapse.

Neurofascin-155 IgG4 in chronic inflammatory demyelinating polyneuropathy.

OBJECTIVE: We report the clinical and serologic features of Japanese patients with chronic inflammatory demyelinating polyneuropathy (CIDP) displaying anti-neurofascin-155 (NF155) immunoglobulin G4 (IgG4) antibodies. METHODS: In sera from 533 patients with CIDP, anti-NF155 IgG4 antibodies were detected by ELISA. Binding of IgG antibodies to central and peripheral nerves was tested. RESULTS: Anti-NF155 IgG4 antibodies were identified in 38 patients (7%) with CIDP, but not in disease controls or normal participants. These patients were younger at onset as compared to 100 anti-NF155-negative patients with CIDP. Twenty-eight patients (74%) presented with sensory ataxia, 16 (42%) showed tremor, 5 (13%) presented with cerebellar ataxia associated with nystagmus, 3 (8%) had demyelinating lesions in the CNS, and 20 of 25 (80%) had poor response to IV immunoglobulin. The clinical features of the antibody-positive patients were statistically more frequent as compared to negative patients with CIDP (n = 100). Anti-NF155 IgG antibodies targeted similarly central and peripheral paranodes. CONCLUSION: Anti-NF155 IgG4 antibodies were associated with a subgroup of patients with CIDP showing a younger age at onset, ataxia, tremor, CNS demyelination, and a poor response to IV immunoglobulin. The autoantibodies may serve as a biomarker to improve patients' diagnosis and guide treatments.

A recurrent KCNQ2 pore mutation causing early onset epileptic encephalopathy has a moderate effect on M current but alters subcellular localization of Kv7 channels.

Mutations in the KCNQ2 gene encoding the voltage-dependent potassium M channel Kv7.2 subunit cause either benign epilepsy or early onset epileptic encephalopathy (EOEE). It has been proposed that the disease severity rests on the inhibitory impact of mutations on M current density. Here, we have analyzed the phenotype of 7 patients carrying the p.A294V mutation located on the S6 segment of the Kv7.2 pore domain (Kv7.2(A294V)). We investigated the functional and subcellular consequences of this mutation and compared it to another mutation (Kv7.2(A294G)) associated with a benign epilepsy and affecting the same residue. We report that all the patients carrying the p.A294V mutation presented the clinical and EEG characteristics of EOEE. In CHO cells, the total expression of Kv7.2(A294V) alone, assessed by western blotting, was only 20% compared to wild-type. No measurable current was recorded in CHO cells expressing Kv7.2(A294V) channel alone. Although the total Kv7.2(A294V) expression was rescued to wild-type levels in cells co-expressing the Kv7.3 subunit, the global current density was still reduced by 83% compared to wild-type heteromeric channel. In a configuration mimicking the patients' heterozygous genotype i.e., Kv7.2(A294V)/Kv7.2/Kv7.3, the global current density was reduced by 30%. In contrast to Kv7.2(A294V), the current density of homomeric Kv7.2(A294G) was not significantly changed compared to wild-type Kv7.2. However, the current density of Kv7.2(A294G)/Kv7.2/Kv7.3 and Kv7.2(A294G)/Kv7.3 channels were reduced by 30% and 50% respectively, compared to wild-type Kv7.2/Kv7.3. In neurons, the p.A294V mutation induced a mislocalization of heteromeric mutant channels to the somato-dendritic compartment, while the p.A294G mutation did not affect the localization of the heteromeric channels to the axon initial segment. We conclude that this position is a hotspot of mutation that can give rise to a severe or a benign epilepsy. The p.A294V mutation does not exert a dominant-negative effect on wild-type subunits but alters the preferential axonal targeting of heteromeric Kv7 channels. Our data suggest that the disease severity is not necessarily a consequence of a strong inhibition of M current and that additional mechanisms such as abnormal subcellular distribution of Kv7 channels could be determinant.

Contactin 1 IgG4 associates to chronic inflammatory demyelinating polyneuropathy with sensory ataxia.

A Spanish group recently reported that four patients with chronic inflammatory demyelinating polyneuropathy carrying IgG4 autoantibodies against contactin 1 showed aggressive symptom onset and poor response to intravenous immunoglobulin. We aimed to describe the clinical and serological features of Japanese chronic inflammatory demyelinating polyneuropathy patients displaying the anti-contactin 1 antibodies. Thirteen of 533 (2.4%) patients with chronic inflammatory demyelinating polyneuropathy had anti-contactin 1 IgG4 whereas neither patients from disease or normal control subjects did (P = 0.02). Three of 13 (23%) patients showed subacute symptom onset, but all of the patients presented with sensory ataxia. Six of 10 (60%) anti-contactin 1 antibody-positive patients had poor response to intravenous immunoglobulin, whereas 8 of 11 (73%) antibody-positive patients had good response to corticosteroids. Anti-contactin 1 IgG4 antibodies are a possible biomarker to guide treatment option.

Calmodulin orchestrates the heteromeric assembly and the trafficking of KCNQ2/3 (Kv7.2/3) channels in neurons.

Mutations in KCNQ2 and KCNQ3 genes are responsible for benign familial neonatal seizures and epileptic encephalopathies. Some of these mutations have been shown to alter the binding of calmodulin (CaM) to specific C-terminal motifs of KCNQ subunits, known as the A and B helices. Here, we show that the mutation I342A in the A helix of KCNQ3 abolishes CaM interaction and strongly decreases the heteromeric association with KCNQ2. The assembly of KCNQ2 with KCNQ3 is essential for their expression at the axon initial segment (AIS). We find that the I342A mutation alters the targeting of KCNQ2/3 subunits at the AIS. However, the traffic of the mutant channels was rescued by provision of exogenous CaM. We show that CaM enhances the heteromeric association of KCNQ2/KCNQ3-I342A subunits by binding to their B helices in a calcium-dependent manner. To further assert the implication of CaM in channel assembly, we inserted a mutation in the second coil-coil domain of KCNQ2 (KCNQ2-L638P) to prevent its heteromerization with KCNQ3. We observe that the expression of a Ca(2+)-insensitive form of CaM favours the assembly of KCNQ3 with KCNQ2-L638P. Our data thus indicate that both apoCaM and Ca(2+)/CaM bind to the C-terminal domains of KCNQ2 and KCNQ3 subunits, and regulate their heteromeric assembly. Hence, CaM may control the composition and distribution of KCNQ channels in neurons.

Neuro-glial interactions at the nodes of Ranvier: implication in health and diseases.

Specific cell adhesion molecules (CAMs) are dedicated to the formation of axo-glial contacts at the nodes of Ranvier of myelinated axons. They play a central role in the organization and maintenance of the axonal domains: the node, paranode, and juxtaparanode. In particular, CAMs are essential for the accumulation of voltage-gated sodium channels at the nodal gap that ensures the rapid and saltatory propagation of the action potentials (APs). The mechanisms regulating node formation are distinct in the central and peripheral nervous systems, and recent studies have highlighted the relative contribution of paranodal junctions and nodal extracellular matrix. In addition, CAMs at the juxtaparanodal domains mediate the clustering of voltage-gated potassium channels which regulate the axonal excitability. In several human pathologies, the axo-glial contacts are altered leading to disruption of the nodes of Ranvier or mis-localization of the ion channels along the axons. Node alterations and the failure of APs to propagate correctly from nodes to nodes along the axons both contribute to the disabilities in demyelinating diseases. This article reviews the mechanisms regulating the association of the axo-glial complexes and the role of CAMs in inherited and acquired neurological diseases.

Antibodies to gliomedin cause peripheral demyelinating neuropathy and the dismantling of the nodes of Ranvier.

Guillain-Barré syndrome (GBS) and chronic inflammatory demyelinating polyneuropathy (CIDP) are conditions that affect peripheral nerves. The mechanisms that underlie demyelination in these neuropathies are unknown. Recently, we demonstrated that the node of Ranvier is the primary site of the immune attack in patients with GBS and CIDP. In particular, GBS patients have antibodies against gliomedin and neurofascin, two adhesion molecules that play a crucial role in the formation of nodes of Ranvier. We demonstrate that immunity toward gliomedin, but not neurofascin, induced a progressive neuropathy in Lewis rats characterized by conduction defects and demyelination in spinal nerves. The clinical symptoms closely followed the titers of anti-gliomedin IgG and were associated with an important deposition of IgG at nodes. Furthermore, passive transfer of antigliomedin IgG induced a severe demyelinating condition and conduction loss. In both active and passive models, the immune attack at nodes occasioned the loss of the nodal clusters for gliomedin, neurofascin-186, and voltage-gated sodium channels. These results indicate that primary immune reaction against gliomedin, a peripheral nervous system adhesion molecule, can be responsible for the initiation or progression of the demyelinating form of GBS. Furthermore, these autoantibodies affect saltatory propagation by dismantling nodal organization and sodium channel clusters. Antibodies reactive against nodal adhesion molecules thus likely participate in the pathologic process of GBS and CIDP.

Nodal proteins are target antigens in Guillain-Barré syndrome.

Neurofascin-186 (NF186), neuronal cell adhesion molecule (NrCAM), and gliomedin are adhesion molecules playing a central role in the formation of nodes of Ranvier. In Guillain-Barré syndrome (GBS), immune attack toward the nodes may participate in the disabilities. Autoantibodies to NF186 and gliomedin have been detected in a rat model of GBS. Here, we investigated the prevalence of antibodies against nodal adhesion molecules in patients with GBS or chronic inflammatory demyelinating polyneuropathy (CIDP). Sera from 100 GBS patients, 50 CIDP patients, 80 disease controls, and 50 healthy controls were tested for their ability to bind the nodes of Ranvier. To characterize the antigens, we performed cell binding assays against NF186, gliomedin, contactin, and NrCAM. We found that 43% of patients with GBS and 30% of patients with CIDP showed IgG fixation at nodes or paranodes. In eight patients with GBS or CIDP, we identified that IgG antibodies recognized the native extracellular domain of NF186, gliomedin, or contactin. Also, 29 patients showed IgM against nodal adhesion molecules. However, we did not detect IgM fixation at nodes or paranodes. Antibodies to gliomedin or NF186 were mostly detected in demyelinating and axonal GBS, respectively. The adsorption of the antibodies to their soluble antigens abolished IgG deposition at nodes and paranodes in nerves, indicating these were specific to NF186, gliomedin, and contactin. In conclusion, gliomedin, NF186, and contactin are novel target antigens in GBS. At nodes, additional epitopes are also the targets of IgG. These results suggest that antibody attack against nodal antigens participates in the etiology of GBS.

Fibronectin type III-like domains of neurofascin-186 protein mediate gliomedin binding and its clustering at the developing nodes of Ranvier.

The cell adhesion molecules (CAMs) of the immunoglobulin superfamily (Ig-CAMs) play a crucial role in the organization of the node of Ranvier in myelinated axons. In the peripheral nervous system, Gliomedin (Gldn) secreted by Schwann cell microvilli binds NgCAM-related CAM (NrCAM) and Neurofascin-186 (NF186) and direct the nodal clustering of voltage-gated sodium channels (Nav). NF186 is the single axonal Gldn partner to ensure Nav clustering at nodes, whereas NrCAM is only required in glial cells (Feinberg, K., Eshed-Eisenbach, Y., Frechter, S., Amor, V., Salomon, D., Sabanay, H., Dupree, J. L., Grumet, M., Brophy, P. J., Shrager, P., and Peles, E. (2010) Neuron 65, 490-502). The olfactomedin domain of Gldn is implicated in the interaction with nodal Ig-CAMs. However, the interacting modules of NrCAM or NF186 involved in Gldn association are unknown. Here, we report that fibronectin type III-like (FnIII) domains of both Ig-CAMs mediate their interaction with Gldn in pulldown and cell binding assays. Using surface plasmon resonance assays, we determined that NrCAM and NF186 display similar affinity constant for their association with Gldn (K(D) of 0.9 and 5.7 nm, respectively). We characterized the FnIII domains 1 and 2 of NF186 as interacting modules that ensure association with Gldn. We found that the soluble FnIII domains of NF186 (FnIII-Fc) bind on Schwann cells and inhibit Gldn and Nav clustering at heminodes, the precursors of mature nodes in myelinating cultures. Our study reveals the unexpected importance of FnIII domains of Ig-CAMs in the organization of nodes of Ranvier in peripheral axons. Thus, NF186 utilizes distinct modules to organize the multimeric nodal complex.

The C-terminal domain of ßIV-spectrin is crucial for KCNQ2 aggregation and excitability at nodes of Ranvier.

The spectrin cytoskeleton has an important function in the targeting of proteins to excitable membrane domains. In axons, ßIV-spectrin stabilizes voltage-gated sodium (Nav) channel clusters at nodes of Ranvier and axon initial segments, two regions crucial for the generation and conduction of action potentials. Here, I investigated the physiology of the neuromuscular junction and peripheral nerves in quivering-3J mice, which show a frame-shift base insertion in the Spnb4 gene and lack the C-terminus of ßIV-spectrin. The quivering-3J mice show prominent spontaneous and evoked hyperactivities at diaphragm neuromuscular junctions. These neuromyotonic and myokymic discharges were more prominent in adult animals when tremors and ataxia were pronounced. Recordings of sciatic and phrenic nerves showed that the hyperactivities originate in myelinated axons distally from nerve terminals. Axon and myelin structure in the PNS were unaffected in quivering-3J mice. Of interest, KCNQ2 subunit aggregates were undetectable at PNS and CNS nodes, whereas Nav and Kv1.1/Kv1.2 channels were properly concentrated at nodal and juxtaparanodal regions, respectively. The protein level of KCNQ2 subunits was normal in mutant animals, suggesting that KCNQ2 subunit absence stems from clustering or trafficking defects in axons. The quivering-3J nodes also presented high densities of ankyrin-G and CK2α, two cytosolic molecules involved with aggregating Nav and KCNQ2/3 channels in axons. Because ßIV-spectrin does not interact with KCNQ2/3 subunits, it is suspected that ßIV-spectrin regulates the distribution of KCNQ2/3 subunits in axonal subdomains via regulatory partners. Retigabine, an activator of KCNQ2/3 channels, attenuated the repetitive activities in quivering-3J mice, suggesting that depletion of KCNQ2 subunits at nodes initiates neuromyotonic/myokymic discharges. These findings demonstrate that spectrin cytoskeleton finely regulates ion channel distribution and implicates KCNQ2/3 subunits in axonal excitability and in myokymia aetiology.

Disruption of neurofascin and gliomedin at nodes of Ranvier precedes demyelination in experimental allergic neuritis.

High densities of voltage-gated sodium (Nav) channels at nodes of Ranvier enable the rapid regeneration and propagation of the action potentials along myelinated axons. In demyelinating pathologies, myelin alterations lead to conduction slowing and even to conduction block. In order to unravel the mechanisms of conduction failure in inflammatory demyelinating diseases, we have examined two models of Guillain-Barré syndrome: the experimental allergic neuritis induced in the Lewis rat by immunization against peripheral myelin (EAN-PM) and against a neuritogenic P2 peptide (EAN-P2). We found that Nav channel clusters were disrupted at EAN-PM nodes. Neurofascin and gliomedin, two cell adhesion molecules involved with aggregating Nav channels at nodes, were selectively affected prior to demyelination in EAN-PM, indicating that degradation of the axo-glial unit initiated node alteration. This was associated with autoantibodies to neurofascin and gliomedin. Node disruption was, however, independent from complement deposition at nodes, and deposits of the terminal complement complex (C5b-9) were found on the external surface of Schwann cells in EAN-PM. In these animals, the paranodal junctions were also affected and Kv1 channels, which are normally juxtaparanodal, were found dispersed at nodes and paranodes. Altogether, these alterations were associated with conduction deficits in EAN-PM ventral spinal roots. EAN-P2 animals also exhibited inflammatory demyelination, but did not show alteration in nodal clusters or autoantibodies. Our results highlighted the complex mechanisms underlying conduction abnormalities in demyelinating disorders, and unraveled neurofascin and gliomedin as two novel immune targets in experimental allergic neuritis.