Clustering Of Sodium Channels Research Articles

Establishment of a Serum-Free Human iPSC-Derived Model of Peripheral Myelination.

Myelination and the formation of nodes of Ranvier are essential for the rapid conduction of nerve impulses along axons in the peripheral nervous system (PNS). While many animal-based and serum-containing models of peripheral myelination have been developed, these have limited ability when it comes to studying genetic disorders affecting peripheral myelination. We report a fully induced pluripotent stem cell (iPSC)-derived human model of peripheral myelination using Schwann cells (SCs) and motoneurons, cultured in a serum-free medium on patterned and nonpatterned surfaces. Results demonstrated iPSC-derived SC-expressed early growth response protein 2 (Egr2), a key transcription factor for myelination, and after ∼30 days in coculture, hallmark features of myelination, including myelin segment and node of Ranvier formation, were observed. Myelin segments were stained for the myelin basic protein, which surrounded neurofilament-stained motoneuron axons. Clusters of voltage-gated sodium channels flanked by paranodal protein contactin-associated protein 1, indicating node of Ranvier formation, were also observed. High-resolution confocal microscopy allowed for 3D reconstruction and measurement of myelin g-ratios of myelin segments, with an average g-ratio of 0.67, consistent with reported values in the literature, indicating mature myelin segment formation. This iPSC-based model of peripheral myelination provides a platform to investigate numerous PNS diseases, including Charcot-Marie Tooth disorder, Guillian-Barre syndrome, chronic inflammatory demyelinating polyneuropathy, and antimyelin-associated glycoprotein peripheral neuropathy, with the potential for greater translatability to humans for improving the applicability for drug-screening programs.

Macrophages are scavengers for injured myelin in a rabbit model of acute inflammatory demyelinating polyneuropathy.

In acute inflammatory demyelinating polyneuropathy (AIDP), myelin vesiculation mediated by complement activation contributes to nerve injury. Macrophage infiltration of the spinal roots has been demonstrated in AIDP, but its pathological significance remains uncertain. The present study aimed to investigate the role of macrophages in the pathogenic sequence of AIDP. A rabbit model of AIDP was induced by immunization with galactocerebroside. Immunostaining was performed to localize the macrophages and myelin injury. The rabbit developed tetraparesis with electrophysiological and pathological features of peripheral nerve demyelination. Immunostaining demonstrated colocalization of IgG antibodies, complement deposition and myelin injury apart from macrophages. Immunostaining and electron microscopy showed myelin injury preceded macrophage infiltration. There was significant disruption of voltage-gated sodium channel clusters at the nodes of Ranvier in the spinal roots. Macrophages acted may as scavengers to remove myelin debris following complement activation-mediated demyelination in the AIDP rabbit. Lesions at the node of Ranvier contribute to conduction failure and muscle weakness.

Sodium channel endocytosis drives axon initial segment plasticity.

Activity-dependent plasticity of the axon initial segment (AIS) endows neurons with the ability to adapt action potential output to changes in network activity. Action potential initiation at the AIS highly depends on the clustering of voltage-gated sodium channels, but the molecular mechanisms regulating their plasticity remain largely unknown. Here, we developed genetic tools to label endogenous sodium channels and their scaffolding protein, to reveal their nanoscale organization and longitudinally image AIS plasticity in hippocampal neurons in slices and primary cultures. We find that N-methyl-d-aspartate receptor activation causes both long-term synaptic depression and rapid internalization of AIS sodium channels within minutes. The clathrin-mediated endocytosis of sodium channels at the distal AIS increases the threshold for action potential generation. These data reveal a fundamental mechanism for rapid activity-dependent AIS reorganization and suggests that plasticity of intrinsic excitability shares conserved features with synaptic plasticity.

Neuromuscular junction transmission failure in aging and sarcopenia: The nexus of the neurological and muscular systems.

Sarcopenia, or age-related decline in muscle form and function, exerts high personal, societal, and economic burdens when untreated. Integrity and function of the neuromuscular junction (NMJ), as the nexus between the nervous and muscular systems, is critical for input and dependable neural control of muscle force generation. As such, the NMJ has long been a site of keen interest in the context of skeletal muscle function deficits during aging and in the context of sarcopenia. Historically, changes of NMJ morphology during aging have been investigated extensively but primarily in aged rodent models. Aged rodents have consistently shown features of NMJ endplate fragmentation and denervation. Yet, the presence of NMJ changes in older humans remains controversial, and conflicting findings have been reported. This review article describes the physiological processes involved in NMJ transmission, discusses the evidence that supports NMJ transmission failure as a possible contributor to sarcopenia, and speculates on the potential of targeting these defects for therapeutic development. The technical approaches that are available for assessment of NMJ transmission, whether each approach has been applied in the context of aging and sarcopenia, and the associated findings are summarized. Like morphological studies, age-related NMJ transmission deficits have primarily been studied in rodents. In preclinical studies, isolated synaptic electrophysiology recordings of endplate currentsor potentials have been mostly used, and paradoxically, have shown enhancement, rather than failure, with aging. Yet, in vivo assessment of single muscle fiber action potential generation using single fiber electromyography and nerve-stimulated muscle force measurements show evidence of NMJ failure in aged mice and rats. Together these findings suggest that endplate response enhancement may be a compensatory response to post-synaptic mechanisms of NMJ transmission failure in aged rodents. Possible, but underexplored, mechanisms of this failure are discussed including the simplification of post-synaptic folding and altered voltage-gated sodium channel clustering or function. In humans, there is limited clinical data that has selectively investigated single synaptic function in the context of aging. If sarcopenic older adults turn out to exhibit notable impairments in NMJ transmission (this has yet to be examined but based on available evidence appears to be plausible) then these NMJ transmission defects present a well-defined biological mechanism and offer a well-defined pathway for clinical implementation. Investigation of small molecules that are currently available clinically or being testing clinically in other disorders may provide a rapid route for development of interventions for older adults impacted by sarcopenia.

ID: 216598 Effects of Sodium Channel Clustering on Activation Thresholds of Nonmyelinated Axons

It is hypothesized that some neurostimulation therapies, such as dorsal root ganglion stimulation (DRGS), may provide therapeutic benefit by activating nonmyelinated neurons (e.g., C-neurons). However, conventional neurostimulation theory suggests that extracellular electrical stimulation preferentially activates large-diameter myelinated neurons over small-diameter nonmyelinated neurons. Interestingly, a recent study of DRGS in rats demonstrated that the activation thresholds of nonmyelinated C-neurons are only ∼1.5 times greater than the activation thresholds of myelinated Abeta-neurons (Chao et al., 2020). It is possible that certain electrophysiological features of C-type neurons unaccounted for in previous computational models could reduce activation thresholds to ranges comparable to the thresholds of myelinated neurons. For example, Nav1.8 sodium channels, which underlie most of the sodium current during the upstroke of action potentials in some C-neurons, have been shown to be distributed in clusters along the axons of C-type neurons forming so-called “micro-nodes” (Pristerà et al., 2012). In this study, we developed a computational model to investigate the impact of Nav1.8-channel clustering on the threshold for action potential generation in nonmyelinated axons.

Crystal structure of Ankyrin-G in complex with a fragment of Neurofascin reveals binding mechanisms required for integrity of the axon initial segment

The axon initial segment (AIS) has characteristically dense clustering of voltage-gated sodium channels (Nav), cell adhesion molecule Neurofascin 186 (Nfasc), and neuronal scaffold protein Ankyrin-G (AnkG) in neurons, which facilitates generation of an action potential and maintenance of axonal polarity. However, the mechanisms underlying AIS assembly, maintenance, and plasticity remain poorly understood. Here, we report the high-resolution crystal structure of the AnkG ankyrin repeat (ANK repeat) domain in complex with its binding site in the Nfasc cytoplasmic tail that shows, in conjunction with binding affinity assays with serial truncation variants, the molecular basis of AnkG–Nfasc binding. We confirm AnkG interacts with the FIGQY motif in Nfasc, and we identify another region required for their high affinity binding. Our structural analysis revealed that ANK repeats form 4 hydrophobic or hydrophilic layers in the AnkG inner groove that coordinate interactions with essential Nfasc residues, including F1202, E1204, and Y1212. Moreover, we show disruption of the AnkG–Nfasc complex abolishes Nfasc enrichment at the AIS in cultured mouse hippocampal neurons. Finally, our structural and biochemical analysis indicated that L1 syndrome-associated mutations in L1CAM, a member of the L1 immunoglobulin family proteins including Nfasc, L1CAM, NrCAM, and CHL1, compromise binding with ankyrins. Taken together, these results define the mechanisms underlying AnkG–Nfasc complex formation and show that AnkG-dependent clustering of Nfasc is required for AIS integrity.

Loss of \u03b24-spectrin impairs Nav channel clustering at the heminode and temporal fidelity of presynaptic spikes in developing auditory brain

Beta-4 (β4)-spectrin, encoded by the gene Sptbn4, is a cytoskeleton protein found at nodes and the axon initial segments (AIS). Sptbn4 mutations are associated with myopathy, neuropathy, and auditory deficits in humans. Related to auditory dysfunction, however, the expression and roles of β4-spectrin at axon segments along the myelinated axon in the developing auditory brain are not well explored. We found during postnatal development, β4-spectrin is critical for voltage-gated sodium channel (Nav) clustering at the heminode along the nerve terminal, but not for the formation of nodal and AIS structures in the auditory brainstem. Presynaptic terminal recordings in Sptbn4geo mice, β4-spectrin null mice, showed an elevated threshold of action potential and increased failures during action potential train at high-frequency. Sptbn4geo mice exhibited a slower central conduction and showed no startle responses, but had normal cochlear function. Taken together, the lack of β4-spectrin impairs Nav clustering at the heminode along the nerve terminal and the temporal fidelity and reliability of presynaptic spikes, leading to central auditory processing deficits during postnatal development.

Length impairments of the axon initial segment in rodent models of attention-deficit hyperactivity disorder and autism spectrum disorder

The axon initial segment (AIS) is a structural neuronal compartment of the proximal axon that plays key roles in sodium channel clustering, action potential initiation, and signal propagation of neuronal outputs. Mutations in constitutive genes of the AIS, such as ANK3, have been identified in patients with neurodevelopmental disorders. Nevertheless, morphological changes in the AIS in neurodevelopmental disorders have not been characterized. In this study, we investigated the length of the AIS in rodent models of attention-deficit hyperactivity disorder (ADHD) and autism spectrum disorder (ASD). We observed abnormalities in AIS length in both animal models. In ADHD model rodents, we observed shorter AIS length in layer 2/3 (L2/3) neurons of the medial prefrontal cortex (mPFC) and primary somatosensory barrel field (S1BF). Further, we observed shorter AIS length in S1BF L5 neurons. In ASD model mice, we observed shorter AIS length in L2/3 and L5 neurons of the S1BF. These results suggest that impairments in AIS length are common phenomena in neurodevelopmental disorders such as ADHD and ASD and may be conserved across species. Our findings provide novel insight into the potential contribution of the AIS to the pathophysiology and pathogenesis of neurodevelopmental disorders.

Pathogenesis of acute and chronic inflammatory neuropathies

Guillain-Barre syndrome (GBS) is classified into axonal and demyelinating categories. Acute motor axonal neuropathy (AMAN), an axonal subtype, was widely recognized in the 1990's. Whereas in Europe and North America, classical demyelinating GBS is the major subtype, AMAN is found for 30–80% of GBS cases in Asia. Over the past 25 years, major advances have been made in understanding the immunopathogenesis of AMAN; animal model studies have shown that there is good evidence to support an anti-GM1 antibody-mediated immune process, triggered by molecular mimicry between gangliosides of motor axons and the microorganism. Complement-mediated attack leads to disruption of nodal sodium channel clusters, and paranodal myelin detachment that is responsible for axonal dysfunction/degeneration in AMAN. CIDP is currently classified into “typical CIDP” and other variants such as “multifocal CIDP (mononeuropathy multiplex type)” and distal CIDP. Typical CIDP is a classic form, characterized by symmetric proximal and distal muscle weakness and motor-dominant manifestation. In typical CIDP, demyelination predominantly affects the distal nerve terminals and nerve roots, presumably because of the lack of the blood-nerve barrier in these regions, and this could be responsible for the “non-nerve length-dependent” distribution of muscle weakness. These findings suggest an importance of humoral immunity in typical CIDP. In contrast, multifocal CIDP is characterized by multifocal demyelination in the intermediate nerve trunks, suggesting that cellular immunity may be predominantly involved in breakdown of the blood-nerve barrier at the site of conduction block. The understanding of the pathophysiology of each CIDP subtype is important for appropriate immune-treatments.

SY7.2. Electrophysiological features in GBS and CIDP

GBS is currently classified into axonal and demyelinating categories. Acute motor axonal neuropathy (AMAN), an axonal subtype of GBS, was widely recognized in the 1990s. Whereas in Europe and North America, acute inflammatory demyelinating polyneuropathy (AIDP) is considered the major subtype of GBS, in East Asia, and Central and South America, AMAN is found for 30–80% of GBS cases. Over the past 20 years, major advances have been made in understanding the immunopathogenesis and pathophysiology of AMAN. Clinically, AMAN is characterized by pure motor involvement, frequent antecedent Campylobacter jejuni enteritis, and serum anti-ganglioside antibodies. Electrophysiological studies frequently reveal, as well as axonal degeneration, rapidly reversible nerve conduction block/slowing (resolved within days to a few weeks); the time-course suggests functional or microstructural changes at the nodes of Ranvier. It is now established that disrupted nodal sodium channel clusters, and paranodal myelin detachment is responsible for conduction block in AMAN. CIDP is currently classified into “typical CIDP” and other variants such as “multifocal acquired demyelinating sensory and motor neuropathy (MADSAM)”. Typical CIDP is a classic form, clinically characterized by symmetric proximal and distal muscle weakness and motor- dominant manifestation. In typical CIDP, demyelination predominantly affects the distal nerve terminals and nerve roots, presumably because of the lack of the blood-nerve barrier in these regions, and this could be responsible for the “non-nerve length-dependent” distribution of muscle weakness. These findings suggest an importance of humoral immunity in typical CIDP. In contrast, MADSAM is characterized by multifocal demyelination in the intermediate nerve trunks. In MADSAM neuropathy, cellular immunity may be predominantly involved in breakdown of the blood-nerve barrier at the site of conduction block. Among the CIDP spectrum, typical CIDP and MADSAM are the major subtypes, and their pathophysiology appears to be distinct. The understanding of the pathophysiology of each CIDP subtype is important for appropriate immune treatments.

A NMR Study of Sodium/Potassium Pumping System in the Node of Ranvier Myelin-Sheath

The QM/MM calculation has been applied to generalize the node of Ranvier results for computing action potentials and electrochemical behavior of membranes that agree with clusters of voltage-gated ion sodium and potassium channels. Ranvier complexes' node is an accurate organization of membrane-bound aqueous boxes. The model applied here shows an electrophysiological phenomenon with simulated structural and physiological data. The quantum effects of various thicknesses in a selected membrane of Galc /DMPC and Galc/NPGS have also been specifically investigated. This allows introducing a capacitive susceptibility that can resonate with the self-induction of helical coils or ion channels, the resonance of which is the main reason for various biological pulses.

Structural and Functional Characterization of a Nav1.5-Mitochondrial Couplon.

The cardiac sodium channel NaV1.5 has a fundamental role in excitability and conduction. Previous studies have shown that sodium channels cluster together in specific cellular subdomains. Their association with intracellular organelles in defined regions of the myocytes, and the functional consequences of that association, remain to be defined. To characterize a subcellular domain formed by sodium channel clusters in the crest region of the myocytes and the subjacent subsarcolemmal mitochondria. Through a combination of imaging approaches including super-resolution microscopy and electron microscopy we identified, in adult cardiac myocytes, a NaV1.5 subpopulation in close proximity to subjacent subsarcolemmal mitochondria; we further found that subjacent subsarcolemmal mitochondria preferentially host the mitochondrial NCLX (Na+/Ca2+ exchanger). This anatomic proximity led us to investigate functional changes in mitochondria resulting from sodium channel activity. Upon TTX (tetrodotoxin) exposure, mitochondria near NaV1.5 channels accumulated more Ca2+ and showed increased reactive oxygen species production when compared with interfibrillar mitochondria. Finally, crosstalk between NaV1.5 channels and mitochondria was analyzed at a transcriptional level. We found that SCN5A (encoding NaV1.5) and SLC8B1 (which encode NaV1.5 and NCLX, respectively) are negatively correlated both in a human transcriptome data set (Genotype-Tissue Expression) and in human-induced pluripotent stem cell-derived cardiac myocytes deficient in SCN5A. We describe an anatomic hub (a couplon) formed by sodium channel clusters and subjacent subsarcolemmal mitochondria. Preferential localization of NCLX to this domain allows for functional coupling where the extrusion of Ca2+ from the mitochondria is powered, at least in part, by the entry of sodium through NaV1.5 channels. These results provide a novel entry-point into a mechanistic understanding of the intersection between electrical and structural functions of the heart.

Myelination and Node of Ranvier Formation in a Human Motoneuron-Schwann Cell Serum-Free Coculture.

Myelination and node of Ranvier formation play an important role in the rapid conduction of nerve impulses, referred to as saltatory conduction, along axons in the peripheral nervous system. We report a human-human myelination model using human primary Schwann cells (SCs) and human-induced pluripotent stem-cell-derived motoneurons utilizing a serum-free medium supplemented with ascorbate to induce myelination, where 41.6% of SCs expressed the master transcription factor for myelination, early growth response protein 2. After 30 days in coculture, myelin segments were visualized using immunocytochemistry for myelin basic protein surrounding neurofilament-stained motor neuron axons, which was confirmed via 3D confocal Raman microscopy, a viable alternative for transmission electron microscopy analysis. The myelination efficiency was 65%, and clusters of voltage-gated sodium channels and the paranodal protein contactin-associated protein 1 indicated node of Ranvier formation. This model has applications to study remyelination and demyelinating diseases, including Charcot-Marie Tooth disorder, Guillian-Barre syndrome, and anti-myelin-associated glycoprotein peripheral neuropathy.

Bio-capacitor consist of insulated myelin-sheath and uninsulated node of Ranvier: a bio-nano-antenna

Myelin consists of fatty molecules (lipids) which are located in the CNC (central nervous system) and as an insulator around nerve cell axons increases the velocities information to transit from one nerve cell to another tissue like an electrical wire (the axon) with insulating material (myelin) around it. Each axon contains multiple long myelinated parts separated from each other through short gaps called “Nodes of Ranvier” or myelin-sheath gaps. A computational model is presented for the simulation of propagated electromagnetic waves in a critical point between insulated myelin-sheath towards uninsulated node of Ranvier. The QM/MM calculation has been applied for generalizing the node of Ranvier results for computing action potentials and electro chemical behavior of membranes which agree with clusters of voltage-gated ion sodium and potassium channels. The node of Ranvier complexes is an accurate organization of membrane-bound aqueous compartments, and the model presented here represents electrophysiological events with combined realistic structural and physiological data. The quantum effects of different thicknesses in the mixed membranes of GalC/DPPC, have also been specifically investigated. It is shown that quantum effects can appear in a small region of free spaces within the membrane thickness due to the number and type of lipid’s layers. In addition, from the view point of quantum effects by Heisenberg rule, it is shown that quantum tunneling is allowed in some micro positions of membrane capacitor systems, while it is forbidden in other forms.

The clustering of voltage-gated sodium channels in various excitable membranes.

In excitable membranes, the clustering of voltage-gated sodium channels (VGSC) serves to enhance excitability at critical sites. The two most profoundly studied sites of channel clustering are the axon initial segment, where action potentials are generated and the node of Ranvier, where action potentials propagate along myelinated axons. The clustering of VGSC is found, however, in other highly excitable sites such as axonal terminals, postsynaptic membranes of dendrites and muscle fibers, and pre-myelinated axons. In this review, different examples of axonal as well as non-axonal clustering of VGSC are discussed and the underlying mechanisms are compared. Whether the clustering of channels is intrinsically or extrinsically induced, it depends on the submembranous actin-based cytoskeleton that organizes these highly specialized membrane microdomains through specific adaptor proteins.

Role of a Contactin multi-molecular complex secreted by oligodendrocytes in nodal protein clustering in the CNS.

The fast and reliable propagation of action potentials along myelinated fibers relies on the clustering of voltage‐gated sodium channels at nodes of Ranvier. Axo‐glial communication is required for assembly of nodal proteins in the central nervous system, yet the underlying mechanisms remain poorly understood. Oligodendrocytes are known to support node of Ranvier assembly through paranodal junction formation. In addition, the formation of early nodal protein clusters (or prenodes) along axons prior to myelination has been reported, and can be induced by oligodendrocyte conditioned medium (OCM). Our recent work on cultured hippocampal neurons showed that OCM‐induced prenodes are associated with an increased conduction velocity (Freeman et al., 2015). We here unravel the nature of the oligodendroglial secreted factors. Mass spectrometry analysis of OCM identified several candidate proteins (i.e., Contactin‐1, ChL1, NrCAM, Noelin2, RPTP/Phosphacan, and Tenascin‐R). We show that Contactin‐1 combined with RPTP/Phosphacan or Tenascin‐R induces clusters of nodal proteins along hippocampal GABAergic axons. Furthermore, Contactin‐1‐immunodepleted OCM or OCM from Cntn1‐null mice display significantly reduced clustering activity, that is restored by addition of soluble Contactin‐1. Altogether, our results identify Contactin‐1 secreted by oligodendrocytes as a novel factor that may influence early steps of nodal sodium channel cluster formation along specific axon populations.

Sodium channel clusters: harmonizing the cardiac conduction orchestra.

Sodium channel clusters: harmonizing the cardiac conduction orchestra.

Postnatal Loss of Neuronal and Glial Neurofascins Differentially Affects Node of Ranvier Maintenance and Myelinated Axon Function.

Intricate molecular interactions between neurons and glial cells underlie the creation of unique domains that are essential for saltatory conduction of action potentials by myelinated axons. Previously, the cell surface adhesion molecule Neurofascin (Nfasc) has been shown to have a dual-role in the establishment of axonal domains from both the glial and neuronal interface. While the neuron-specific isoform of Neurofascin (NF186) is indispensable for clustering of voltage-gated sodium channels at nodes of Ranvier; the glial-specific isoform of Neurofascin (NF155) is required for myelinating glial cells to organize the paranodal domain. Although many studies have addressed the individual roles of NF155 and NF186 in assembling paranodes and nodes, respectively; critical questions about their roles in the maintenance and long-term health of the myelinated axons remain, which we aimed to address in these studies. Here using spatiotemporal ablation of Neurofascin in neurons alone or together with myelinating glia, we report that loss of NF186 individually from postnatal mice leads to progressive nodal destabilization and axonal degeneration. While individual ablation of paranodal NF155 does not disrupt nodes of Ranvier; loss of NF186 combined with NF155 causes more accelerated nodal destabilization than loss of NF186 alone, providing strong evidence regarding a supporting role for paranodes in nodal maintenance. In both cases of NF186 loss, myelinating axons show ultrastructural changes and degeneration. Our studies reveal that long-term maintenance of nodes and ultimately the health of axons is correlated with the stability of NF186 within the nodal complex and the presence of auxiliary paranodes.

Neuronal Ndrg4 Is Essential for Nodes of Ranvier Organization in Zebrafish.

Axon ensheathment by specialized glial cells is an important process for fast propagation of action potentials. The rapid electrical conduction along myelinated axons is mainly due to its saltatory nature characterized by the accumulation of ion channels at the nodes of Ranvier. However, how these ion channels are transported and anchored along axons is not fully understood. We have identified N-myc downstream-regulated gene 4, ndrg4, as a novel factor that regulates sodium channel clustering in zebrafish. Analysis of chimeric larvae indicates that ndrg4 functions autonomously within neurons for sodium channel clustering at the nodes. Molecular analysis of ndrg4 mutants shows that expression of snap25 and nsf are sharply decreased, revealing a role of ndrg4 in controlling vesicle exocytosis. This uncovers a previously unknown function of ndrg4 in regulating vesicle docking and nodes of Ranvier organization, at least through its ability to finely tune the expression of the t-SNARE/NSF machinery.

Activity-dependent formation and location of voltage-gated sodium channel clusters at a CNS nerve terminal during postnatal development.

Clustering of voltage-gated sodium (Nav) channels and their distribution along the axon, specifically at the unmyelinated axon segment next to the nerve terminal, are essential for tuning propagated action potentials. Nav channel clusters near the nerve terminal and their location as a function of neuronal position along the mediolateral axis are controlled by auditory inputs after hearing onset. Thus sound-mediated neuronal activity influences the tonotopic organization of Nav channels at the nerve terminal in the auditory brain stem.