Mechanisms of node of Ranvier assembly.

The nodes of Ranvier have clustered Na+ and K+ channels necessary for rapid and efficient axonal action potential conduction. However, detailed mechanisms of channel clustering have only recently been identified: they include two independent axon-glia interactions that converge on distinct axonal cytoskeletons. Here, we discuss how glial cell adhesion molecules and the extracellular matrix molecules that bind them assemble combinations of ankyrins, spectrins and other cytoskeletal scaffolding proteins, which cluster ion channels. We present a detailed molecular model, incorporating these overlapping mechanisms, to explain how the nodes of Ranvier are assembled in both the peripheral and central nervous systems.

Absence of CNTNAP2 leads to epilepsy, neuronal migration abnormalities, and core autism-related deficits.

Although many genes predisposing to autism spectrum disorders (ASD) have been identified, the biological mechanism(s) remain unclear. Mouse models based on human disease-causing mutations provide the potential for understanding gene function and novel treatment development. Here, we characterize a mouse knockout of the Cntnap2 gene, which is strongly associated with ASD and allied neurodevelopmental disorders. Cntnap2(-/-) mice show deficits in the three core ASD behavioral domains, as well as hyperactivity and epileptic seizures, as have been reported in humans with CNTNAP2 mutations. Neuropathological and physiological analyses of these mice before the onset of seizures reveal neuronal migration abnormalities, reduced number of interneurons, and abnormal neuronal network activity. In addition, treatment with the FDA-approved drug risperidone ameliorates the targeted repetitive behaviors in the mutant mice. These data demonstrate a functional role for CNTNAP2 in brain development and provide a new tool for mechanistic and therapeutic research in ASD.

Complement-membrane regulatory proteins are absent from the nodes of Ranvier in the peripheral nervous system.

BACKGROUND: Homozygous CD59-deficient patients manifest with recurrent peripheral neuropathy resembling Guillain-Barré syndrome (GBS), hemolytic anemia and recurrent strokes. Variable mutations in CD59 leading to loss of function have been described and, overall, 17/18 of patients with any mutation presented with recurrent GBS. Here we determine the localization and possible role of membrane-bound complement regulators, including CD59, in the peripheral nervous systems (PNS) of mice and humans. METHODS: We examined the localization of membrane-bound complement regulators in the peripheral nerves of healthy humans and a CD59-deficient patient, as well as in wild-type (WT) and CD59a-deficient mice. Cross sections of teased sciatic nerves and myelinating dorsal root ganglia (DRG) neuron/Schwann cell cultures were examined by confocal and electron microscopy. RESULTS: We demonstrate that CD59a-deficient mice display normal peripheral nerve morphology but develop myelin abnormalities in older age. They normally express myelin protein zero (P0), ankyrin G (AnkG), Caspr, dystroglycan, and neurofascin. Immunolabeling of WT nerves using antibodies to CD59 and myelin basic protein (MBP), P0, and AnkG revealed that CD59 was localized along the internode but was absent from the nodes of Ranvier. CD59 was also detected in blood vessels within the nerve. Finally, we show that the nodes of Ranvier lack other complement-membrane regulatory proteins, including CD46, CD55, CD35, and CR1-related gene-y (Crry), rendering this area highly exposed to complement attack. CONCLUSION: The Nodes of Ranvier lack CD59 and are hence not protected from complement terminal attack. The myelin unit in human PNS is protected by CD59 and CD55, but not by CD46 or CD35. This renders the nodes and myelin in the PNS vulnerable to complement attack and demyelination in autoinflammatory Guillain-Barré syndrome, as seen in CD59 deficiency.

Nodes of Ranvier in health and disease.

Action potential propagation along myelinated axons depends on the geometry of the myelin unit and the division of the underlying axon to specialized domains. The latter include the nodes of Ranvier (NOR), the paranodal junction (PNJ) flanking the nodes, and the adjacent juxtaparanodal region that is located below the compact myelin of the internode. Each of these domains contains a unique composition of axoglial adhesion molecules (CAMs) and cytoskeletal scaffolding proteins, which together direct the placement of specific ion channels at the nodal and juxtaparanodal axolemma. In the last decade it has become increasingly clear that antibodies to some of these axoglial CAMs cause immune-mediated neuropathies. In the current review we detail the molecular composition of the NOR and adjacent membrane domains, describe the function of different CAM complexes that mediate axon-glia interactions along the myelin unit, and discuss their involvement and the underlying mechanisms taking place in peripheral nerve pathologies. This growing group of pathologies represent a new type of neuropathies termed "nodopathies" or "paranodopathies" that are characterized by unique clinical and molecular features which together reflect the mechanisms underlying the molecular assembly and maintenance of this specialized membrane domain.

Differential Contribution of Cadm1-Cadm3 Cell Adhesion Molecules to Peripheral Myelinated Axons.

Cell adhesion proteins of the Cadm (SynCAM/Necl) family regulate myelination and the organization of myelinated axons. In the peripheral nervous system (PNS), intercellular contact between Schwann cells and their underlying axons is believed to be mediated by binding of glial Cadm4 to axonal Cadm3 or Cadm2. Nevertheless, given that distinct neurons express different combinations of the Cadm proteins, the identity of the functional axonal ligand for Cadm4 remains to be determined. Here, we took a genetic approach to compare the phenotype of Cadm4 null mice, which exhibit abnormal distribution of Caspr and Kv1 potassium channels, with mice lacking different combinations of Cadm1-Cadm3 genes. We show that in contrast to mice lacking the single Cadm1, Cadm2, or Cadm3 genes, genetic ablation of all three phenocopies the abnormalities detected in the absence of Cadm4. Similar defects were observed in double mutant mice lacking Cadm3 and Cadm2 (i.e., Cadm3-/-/Cadm2-/-) or Cadm3 and Cadm1 (i.e., Cadm3-/-/Cadm1-/-), but not in mice lacking Cadm1 and Cadm2 (i.e., Cadm1-/-/Cadm2-/-). Furthermore, axonal organization abnormalities were also detected in Cadm3 null mice that were heterozygous for the two other axonal Cadms. Our results identify Cadm3 as the main axonal ligand for glial Cadm4, and reveal that its absence could be compensated by the combined action of Cadm2 and Cadm1.SIGNIFICANCE STATEMENT Myelination by Schwann cells enables fast conduction of action potentials along motor and sensory axons. In these nerves, Schwann cell-axon contact is mediated by cell adhesion molecules of the Cadm family. Cadm4 in Schwann cells regulates axonal ensheathment and myelin wrapping, as well as the organization of the axonal membrane, but the identity of its axonal ligands is not clear. Here, we reveal that Cadm mediated axon-glia interactions depend on a hierarchical adhesion code that involves multiple family members. Our results provide important insights into the molecular mechanisms of axon-glia communication, and the function of Cadm proteins in PNS myelin.

A CADM3 variant causes Charcot-Marie-Tooth disease with marked upper limb involvement.

The CADM family of proteins consists of four neuronal specific adhesion molecules (CADM1, CADM2, CADM3 and CADM4) that mediate the direct contact and interaction between axons and glia. In the peripheral nerve, axon-Schwann cell interaction is essential for the structural organization of myelinated fibres and is primarily mediated by the binding of CADM3, expressed in axons, to CADM4, expressed by myelinating Schwann cells. We have identified-by whole exome sequencing-three unrelated families, including one de novo patient, with axonal Charcot-Marie-Tooth disease (CMT2) sharing the same private variant in CADM3, Tyr172Cys. This variant is absent in 230 000 control chromosomes from gnomAD and predicted to be pathogenic. Most CADM3 patients share a similar phenotype consisting of autosomal dominant CMT2 with marked upper limb involvement. High resolution mass spectrometry analysis detected a newly created disulphide bond in the mutant CADM3 potentially modifying the native protein conformation. Our data support a retention of the mutant protein in the endoplasmic reticulum and reduced cell surface expression in vitro. Stochastic optical reconstruction microscopy imaging revealed decreased co-localization of the mutant with CADM4 at intercellular contact sites. Mice carrying the corresponding human mutation (Cadm3Y170C) showed reduced expression of the mutant protein in axons. Cadm3Y170C mice showed normal nerve conduction and myelin morphology, but exhibited abnormal axonal organization, including abnormal distribution of Kv1.2 channels and Caspr along myelinated axons. Our findings indicate the involvement of abnormal axon-glia interaction as a disease-causing mechanism in CMT patients with CADM3 mutations.

Neuronal deletion of Wwox, associated with WOREE syndrome, causes epilepsy and myelin defects.

WWOX-related epileptic encephalopathy (WOREE) syndrome caused by human germline bi-allelic mutations in WWOX is a neurodevelopmental disorder characterized by intractable epilepsy, severe developmental delay, ataxia and premature death at the age of 2-4 years. The underlying mechanisms of WWOX actions are poorly understood. In the current study, we show that specific neuronal deletion of murine Wwox produces phenotypes typical of the Wwox-null mutation leading to brain hyperexcitability, intractable epilepsy, ataxia and postnatal lethality. A significant decrease in transcript levels of genes involved in myelination was observed in mouse cortex and hippocampus. Wwox-mutant mice exhibited reduced maturation of oligodendrocytes, reduced myelinated axons and impaired axonal conductivity. Brain hyperexcitability and hypomyelination were also revealed in human brain organoids with a WWOX deletion. These findings provide cellular and molecular evidence for myelination defects and hyperexcitability in the WOREE syndrome linked to neuronal function of WWOX.

Accumulation of Neurofascin at Nodes of Ranvier Is Regulated by a Paranodal Switch.

The paranodal junctions flank mature nodes of Ranvier and provide a barrier between ion channels at the nodes and juxtaparanodes. These junctions also promote node assembly and maintenance by mechanisms that are poorly understood. Here, we examine their role in the accumulation of NF186, a key adhesion molecule of PNS and CNS nodes. We previously showed that NF186 is initially targeted/accumulates via its ectodomain to forming PNS (hemi)nodes by diffusion trapping, whereas it is later targeted to mature nodes by a transport-dependent mechanism mediated by its cytoplasmic segment. To address the role of the paranodes in this switch, we compared accumulation of NF186 ectodomain and cytoplasmic domain constructs in WT versus paranode defective (i.e., Caspr-null) mice. Both pathways are affected in the paranodal mutants. In the PNS of Caspr-null mice, diffusion trapping mediated by the NF186 ectodomain aberrantly persists into adulthood, whereas the cytoplasmic domain/transport-dependent targeting is impaired. In contrast, accumulation of NF186 at CNS nodes does not undergo a switch; it is predominantly targeted to both forming and mature CNS nodes via its cytoplasmic domain and requires intact paranodes. Fluorescence recovery after photobleaching analysis indicates that the paranodes provide a membrane diffusion barrier that normally precludes diffusion of NF186 to nodes. Linkage of paranodal proteins to the underlying cytoskeleton likely contributes to this diffusion barrier based on 4.1B and ßII spectrin expression in Caspr-null mice. Together, these results implicate the paranodes as membrane diffusion barriers that regulate targeting to nodes and highlight differences in the assembly of PNS and CNS nodes.SIGNIFICANCE STATEMENT Nodes of Ranvier are essential for effective saltatory conduction along myelinated axons. A major question is how the various axonal proteins that comprise the multimeric nodal complex accumulate at this site. Here we examine how targeting of NF186, a key nodal adhesion molecule, is regulated by the flanking paranodal junctions. We show that the transition from diffusion-trapping to transport-dependent accumulation of NF186 requires the paranodal junctions. We also demonstrate that these junctions are a barrier to diffusion of axonal proteins into the node and highlight differences in PNS and CNS node assembly. These results provide new insights into the mechanism of node assembly and the pathophysiology of neurologic disorders in which impaired paranodal function contributes to clinical disability.

N-Wasp Regulates Oligodendrocyte Myelination.

Oligodendrocyte myelination depends on actin cytoskeleton rearrangement. Neural Wiskott-Aldrich syndrome protein(N-Wasp) is an actin nucleation factor that promotes polymerization of branched actin filaments. N-Wasp activity is essential for myelin membrane wrapping by Schwann cells, but its role in oligodendrocytes and CNS myelination remains unknown. Here we report that oligodendrocytes-specific deletion of N-Wasp in mice of both sexes resulted in hypomyelination (i.e., reduced number of myelinated axons and thinner myelin profiles), as well as substantial focal hypermyelination reflected by the formation of remarkably long myelin outfolds. These myelin outfolds surrounded unmyelinated axons, neuronal cell bodies, and other myelin profiles. The latter configuration resulted in pseudo-multimyelin profiles that were often associated with axonal detachment and degeneration throughout the CNS, including in the optic nerve, corpus callosum, and the spinal cord. Furthermore, developmental analysis revealed that myelin abnormalities were already observed during the onset of myelination, suggesting that they are formed by aberrant and misguided elongation of the oligodendrocyte inner lip membrane. Our results demonstrate that N-Wasp is required for the formation of normal myelin in the CNS. They also reveal that N-Wasp plays a distinct role in oligodendrocytes compared with Schwann cells, highlighting a difference in the regulation of actin dynamics during CNS and PNS myelination.SIGNIFICANCE STATEMENT Myelin is critical for the normal function of the nervous system by facilitating fast conduction of action potentials. During the process of myelination in the CNS, oligodendrocytes undergo extensive morphological changes that involve cellular process extension and retraction, axonal ensheathment, and myelin membrane wrapping. Here we present evidence that N-Wasp, a protein regulating actin filament assembly through Arp2/3 complex-dependent actin nucleation, plays a critical role in CNS myelination, and its absence leads to several myelin abnormalities. Our data provide an important step into the understanding of the molecular mechanisms underlying CNS myelination.

Loss of Cntnap2 Causes Axonal Excitability Deficits, Developmental Delay in Cortical Myelination, and Abnormal Stereotyped Motor Behavior.

Contactin-associated protein-like 2 (Caspr2) is found at the nodes of Ranvier and has been associated with physiological properties of white matter conductivity. Genetic variation in CNTNAP2, the gene encoding Caspr2, has been linked to several neurodevelopmental conditions, yet pathophysiological effects of CNTNAP2 mutations on axonal physiology and brain myelination are unknown. Here, we have investigated mouse mutants for Cntnap2 and found profound deficiencies in the clustering of Kv1-family potassium channels in the juxtaparanodes of brain myelinated axons. These deficits are associated with a change in the waveform of axonal action potentials and increases in postsynaptic excitatory responses. We also observed that the normal process of myelination is delayed in Cntnap2 mutant mice. This later phenotype is a likely modulator of the developmental expressivity of the stereotyped motor behaviors that characterize Cntnap2 mutant mice. Altogether, our results reveal a mechanism linked to white matter conductivity through which mutation of CNTNAP2 may affect neurodevelopmental outcomes.

Glial M6B stabilizes the axonal membrane at peripheral nodes of Ranvier.

Glycoprotein M6B and the closely related proteolipid protein regulate oligodendrocyte myelination in the central nervous system, but their role in the peripheral nervous system is less clear. Here we report that M6B is located at nodes of Ranvier in peripheral nerves where it stabilizes the nodal axolemma. We show that M6B is co-localized and associates with gliomedin at Schwann cell microvilli that are attached to the nodes. Developmental analysis of sciatic nerves, as well as of myelinating Schwann cells/dorsal root ganglion neurons cultures, revealed that M6B is already present at heminodes, which are considered the precursors of mature nodes of Ranvier. However, in contrast to gliomedin, which accumulates at heminodes with or prior to Na+ channels, we often detected Na+ channel clusters at heminodes without any associated M6B, indicating that it is not required for initial channel clustering. Consistently, nodal cell adhesion molecules (NF186, NrCAM), ion channels (Nav1.2 and Kv7.2), cytoskeletal proteins (AnkG and ßIV spectrin), and microvilli components (pERM, syndecan3, gliomedin), are all present at both heminodes and mature nodes of Ranvier in Gpm6b null mice. Using transmission electron microscopy, we show that the absence of M6B results in progressive appearance of nodal protrusions of the nodal axolemma, that are often accompanied by the presence of enlarged mitochondria. Our results reveal that M6B is a Schwann cell microvilli component that preserves the structural integrity of peripheral nodes of Ranvier.

Synaptic abnormalities and cytoplasmic glutamate receptor aggregates in contactin associated protein-like 2/Caspr2 knockout neurons.

Central glutamatergic synapses and the molecular pathways that control them are emerging as common substrates in the pathogenesis of mental disorders. Genetic variation in the contactin associated protein-like 2 (CNTNAP2) gene, including copy number variations, exon deletions, truncations, single nucleotide variants, and polymorphisms have been associated with intellectual disability, epilepsy, schizophrenia, language disorders, and autism. CNTNAP2, encoded by Cntnap2, is required for dendritic spine development and its absence causes disease-related phenotypes in mice. However, the mechanisms whereby CNTNAP2 regulates glutamatergic synapses are not known, and cellular phenotypes have not been investigated in Cntnap2 knockout neurons. Here we show that CNTNAP2 is present in dendritic spines, as well as axons and soma. Structured illumination superresolution microscopy reveals closer proximity to excitatory, rather than inhibitory synaptic markers. CNTNAP2 does not promote the formation of synapses and cultured neurons from Cntnap2 knockout mice do not show early defects in axon and dendrite outgrowth, suggesting that CNTNAP2 is not required at this stage. However, mature neurons from knockout mice show reduced spine density and levels of GluA1 subunits of AMPA receptors in spines. Unexpectedly, knockout neurons show large cytoplasmic aggregates of GluA1. Here we characterize, for the first time to our knowledge, synaptic phenotypes in Cntnap2 knockout neurons and reveal a novel role for CNTNAP2 in GluA1 trafficking. Taken together, our findings provide insight into the biological roles of CNTNAP2 and into the pathogenesis of CNTNAP2-associated neuropsychiatric disorders.

The myelin proteolipid plasmolipin forms oligomers and induces liquid-ordered membranes in the Golgi complex.

Myelin comprises a compactly stacked massive surface area of protein-poor thick membrane that insulates axons to allow fast signal propagation. Increasing levels of the myelin protein plasmolipin (PLLP) were correlated with post-natal myelination; however, its function is unknown. Here, the intracellular localization and dynamics of PLLP were characterized in primary glial and cultured cells using fluorescently labeled PLLP and antibodies against PLLP. PLLP localized to and recycled between the plasma membrane and the Golgi complex. In the Golgi complex, PLLP forms oligomers based on fluorescence resonance energy transfer (FRET) analyses. PLLP oligomers blocked Golgi to plasma membrane transport of the secretory protein vesicular stomatitis virus G protein (VSVG), but not of a VSVG mutant with an elongated transmembrane domain. Laurdan staining analysis showed that this block is associated with PLLP-induced proliferation of liquid-ordered membranes. These findings show the capacity of PLLP to assemble potential myelin membrane precursor domains at the Golgi complex through its oligomerization and ability to attract liquid-ordered lipids. These data support a model in which PLLP functions in myelin biogenesis through organization of myelin liquid-ordered membranes in the Golgi complex.

Auto-antibodies to contactin-associated protein 1 (Caspr) in two patients with painful inflammatory neuropathy.

Auto-antibodies against the paranodal proteins neurofascin-155 and contactin-1 have recently been described in patients with chronic inflammatory demyelinating polyradiculoneuropathy and are associated with a distinct clinical phenotype and response to treatment. Contactin-associated protein 1 (Caspr, encoded by CNTNAP1) is a paranodal protein that is attached to neurofascin-155 and contactin-1 (CNTN1) but has not yet been identified as a sole antigen in patients with inflammatory neuropathies. In the present study, we screened a cohort of 35 patients with chronic inflammatory demyelinating polyradiculoneuropathy (age range 20-80, 10 female, 25 male) and 22 patients with Guillain-Barré syndrome (age range 17-86, eight female, 14 male) for autoantibodies against paranodal antigens. We identified two patients, one with chronic inflammatory demyelinating polyradiculoneuropathy and one with Guillain-Barré syndrome, with autoantibodies against Caspr by binding assays using Caspr transfected human embryonic kidney cells and murine teased fibres. IgG3 was the predominant autoantibody subclass in the patient with Guillain-Barré syndrome, IgG4 was predominant in the patient with chronic inflammatory demyelinating polyradiculoneuropathy. Accordingly, complement deposition after binding to HEK293 cells was detectable in the patient with IgG3 autoantibodies only, not in the patient with IgG4. Severe disruption of the paranodal and nodal architecture was detectable in teased fibres of the sural nerve biopsy and in dermal myelinated fibres, supporting the notion of the paranodes being the site of pathology. Deposition of IgG at the paranodes was detected in teased fibre preparations of the sural nerve, further supporting the pathogenicity of anti-Caspr autoantibodies. Pain was one of the predominant findings in both patients, possibly reflected by binding of patients' IgG to TRPV1 immunoreactive dorsal root ganglia neurons. Our results demonstrate that the paranodal protein Caspr constitutes a new antigen that leads to autoantibody generation as part of the novel entity of neuropathies associated with autoantibodies against paranodal proteins.

Expression of Cntnap2 (Caspr2) in multiple levels of sensory systems.

Genome-wide association studies and copy number variation analyses have linked contactin associated protein 2 (Caspr2, gene name Cntnap2) with autism spectrum disorder (ASD). In line with these findings, mice lacking Caspr2 (Cntnap2(-/-)) were shown to have core autism-like deficits including abnormal social behavior and communication, and behavior inflexibility. However the role of Caspr2 in ASD pathogenicity remains unclear. Here we have generated a new Caspr2:tau-LacZ knock-in reporter line (Cntnap2(tlacz/tlacz)), which enabled us to monitor the neuronal circuits in the brain expressing Caspr2. We show that Caspr2 is expressed in many brain regions and produced a comprehensive report of Caspr2 expression. Moreover, we found that Caspr2 marks all sensory modalities: it is expressed in distinct brain regions involved in different sensory processings and is present in all primary sensory organs. Olfaction-based behavioral tests revealed that mice lacking Caspr2 exhibit abnormal response to sensory stimuli and lack preference for novel odors. These results suggest that loss of Caspr2 throughout the sensory system may contribute to the sensory manifestations frequently observed in ASD.

Interaction proteomics of canonical Caspr2 (CNTNAP2) reveals the presence of two Caspr2 isoforms with overlapping interactomes.

Autism is a human developmental brain disorder characterized by impaired social interaction and communication. Contactin-associated protein-like 2 (Caspr2, CNTNAP2) is a known genetic risk factor of autism. However, how this protein might contribute to pathology is unclear. In this study, we demonstrate that Caspr2 is abundantly present in lipid raft and in the synaptic membrane but is highly depleted in the postsynaptic density. The Caspr2 protein level in hippocampus is present at a constant level during synapse formation and myelination from P0 to P84. Interaction proteomics revealed the interactors of Caspr2, including CNTN2, KCNAs, members of the ADAM family (ADAM22, ADAM23 and ADAM11), members of LGI family and MAGUKs (DLGs and MPPs). Interestingly, a short form of Caspr2 was detected, which lacks most of the extracellular domains, however, is still associated with ADAM22 and to a lesser extent LGI1 and Kv1 channels. The comprehensive Caspr2 interactome revealed here might aid in understanding the molecular mechanisms underlying autism. This article is part of a Special Issue titled Neuroproteomics: Applications in Neuroscience and Neurology.

Specific inhibition of secreted NRG1 types I-II by heparin enhances Schwann Cell myelination.

Primary cultures of mixed neuron and Schwann cells prepared from dorsal root ganglia (DRG) are extensively used as a model to study myelination. These dissociated DRG cultures have the particular advantage of bypassing the difficulty in purifying mouse Schwann cells, which is often required when using mutant mice. However, the drawback of this experimental system is that it yields low amounts of myelin. Here we report a simple and efficient method to enhance myelination in vitro. We show that the addition of heparin or low molecular weight heparin to mixed DRG cultures markedly increases Schwann cells myelination. The myelin promoting activity of heparin results from specific inhibition of the soluble immunoglobulin (Ig)-containing isoforms of neuregulin 1 (i.e., NRG1 types I and II) that negatively regulates myelination. Heparin supplement provides a robust and reproducible method to increase myelination in a simple and commonly used culture system. GLIA 2016;64:1227-1234.

Myelin-associated glycoprotein gene mutation causes Pelizaeus-Merzbacher disease-like disorder.

Pelizaeus-Merzbacher disease is an X-linked hypomyelinating leukodystrophy caused by mutations or rearrangements in PLP1. It presents in infancy with nystagmus, jerky head movements, hypotonia and developmental delay evolving into spastic tetraplegia with optic atrophy and variable movement disorders. A clinically similar phenotype caused by recessive mutations in GJC2 is known as Pelizaeus-Merzbacher-like disease. Both genes encode proteins associated with myelin. We describe three siblings of a consanguineous family manifesting the typical infantile-onset Pelizaeus-Merzbacher disease-like phenotype slowly evolving into a form of complicated hereditary spastic paraplegia with mental retardation, dysarthria, optic atrophy and peripheral neuropathy in adulthood. Magnetic resonance imaging and spectroscopy were consistent with a demyelinating leukodystrophy. Using genetic linkage and exome sequencing, we identified a homozygous missense c.399C>G; p.S133R mutation in MAG. This gene, previously associated with hereditary spastic paraplegia, encodes myelin-associated glycoprotein, which is involved in myelin maintenance and glia-axon interaction. This mutation is predicted to destabilize the protein and affect its tertiary structure. Examination of the sural nerve biopsy sample obtained in childhood in the oldest sibling revealed complete absence of myelin-associated glycoprotein accompanied by ill-formed onion-bulb structures and a relatively thin myelin sheath of the affected axons. Immunofluorescence, cell surface labelling, biochemical analysis and mass spectrometry-based proteomics studies in a variety of cell types demonstrated a devastating effect of the mutation on post-translational processing, steady state expression and subcellular localization of myelin-associated glycoprotein. In contrast to the wild-type protein, the p.S133R mutant was retained in the endoplasmic reticulum and was subjected to endoplasmic reticulum-associated protein degradation by the proteasome. Our findings identify involvement of myelin-associated glycoprotein in this family with a disorder affecting the central and peripheral nervous system, and suggest that loss of the protein function is responsible for the unique clinical phenotype.

Caspr and caspr2 are required for both radial and longitudinal organization of myelinated axons.

In myelinated peripheral axons, Kv1 potassium channels are clustered at the juxtaparanodal region and at an internodal line located along the mesaxon and below the Schmidt-Lanterman incisures. This polarized distribution is controlled by Schwann cells and requires specific cell adhesion molecules (CAMs). The accumulation of Kv1 channels at the juxtaparanodal region depends on the presence of Caspr2 at this site, as well as on the presence of Caspr at the adjacent paranodal junction. However, the localization of these channels along the mesaxonal internodal line still persists in the absence of each one of these CAMs. By generating mice lacking both Caspr and Caspr2 (caspr(-/-)/caspr2(-/-)), we now reveal compensatory functions of the two proteins in the organization of the axolemma. Although Kv1 channels are clustered along the inner mesaxon and in a circumferential ring below the incisures in the single mutants, in sciatic nerves of caspr(-/-)/caspr2(-/-) mice, these channels formed large aggregates that were dispersed along the axolemma, demonstrating that internodal localization of Kv1 channels requires either Caspr or Caspr2. Furthermore, deletion of both Caspr and Caspr2 also resulted in widening of the nodes of Ranvier, suggesting that Caspr2 (which is present at paranodes in the absence of Caspr) can partially compensate for the barrier function of Caspr at this site even without the formation of a distinct paranodal junction. Our results indicate that Caspr and Caspr2 are required for the organization of the axolemma both radially, manifested as the mesaxonal line, and longitudinally, demarcated by the nodal domains.