axon-Schwann Cell Interactions Research Articles

Glia trigger endocytic clearance of axonal proteins to promote rodent myelination

Axons undergo striking changes in their content and distribution of cell adhesion molecules (CAMs) and ion channels during myelination that underlies the switch from continuous to saltatory conduction. These changes include the removal of a large cohort of uniformly distributed CAMs that mediate initial axon-Schwann cell interactions and their replacement by a subset of CAMs that mediate domain-specific interactions of myelinated fibers. Here, using rodent models, we examine the mechanisms and significance of this removal of axonal CAMs. We show that Schwann cells just prior to myelination locally activate clathrin-mediated endocytosis (CME) in axons, thereby driving clearance of a broad array of axonal CAMs. CAMs engineeredto resist endocytosis are persistently expressed along the axon and delay both PNS and CNS myelination. Thus, glia non-autonomously activate CME in axons to downregulate axonal CAMs and presumptively axo-glial adhesion. This promotes the transition from ensheathment to myelination while simultaneously sculpting the formation of axonal domains.

Peripheral Nervous System (PNS) Myelin Diseases.

This is a review of inherited and acquired causes of human demyelinating neuropathies and a subset of disorders that affect axon-Schwann cell interactions. Nearly all inherited demyelinating neuropathies are caused by mutations in genes that are expressed by myelinating Schwann cells, affecting diverse functions in a cell-autonomous manner. The most common acquired demyelinating neuropathies are Guillain-Barré syndrome and chronic, inflammatory demyelinating polyneuropathy, both of which are immune-mediated. An additional group of inherited and acquired disorders affect axon-Schwann cell interactions in the nodal region. Overall, these disorders affect the formation of myelin and its maintenance, with superimposed axonal loss that is clinically important.

EBP50 is a key molecule for the Schwann cell-axon interaction in peripheral nerves

Peripheral nerve injury disrupts the Schwann cell-axon interaction and the cellular communication between them. The peripheral nervous system has immense potential for regeneration extensively due to the innate plastic potential of Schwann cells (SCs) that allows SCs to interact with the injured axons and exert specific repair functions essential for peripheral nerve regeneration. In this study, we show that EBP50 is essential for the repair function of SCs and regeneration following nerve injury. The increased expression of EBP50 in the injured sciatic nerve of control mice suggested a significant role in regeneration. The ablation of EBP50 in mice resulted in delayed nerve repair, recovery of behavioral function, and remyelination following nerve injury. EBP50 deficiency led to deficits in SC functions, including proliferation, migration, cytoskeleton dynamics, and axon interactions. The adeno-associated virus (AAV)-mediated local expression of EBP50 improved SCs migration, functional recovery, and remyelination. ErbB2-related proteins were not differentially expressed in EBP50-deficient sciatic nerves following injury. EBP50 binds and stabilizes ErbB2 and activates the repair functions to promote regeneration. Thus, we identified EBP50 as a potent SC protein that can enhance the regeneration and functional recovery driven by NRG1-ErbB2 signaling, as well as a novel regeneration modulator capable of potential therapeutic effects.

First person – Clara Mutschler

ABSTRACT First Person is a series of interviews with the first authors of a selection of papers published in Journal of Cell Science, helping researchers promote themselves alongside their papers. Clara Mutschler is first author on ‘Schwann cells are axo-protective after injury irrespective of myelination status in mouse Schwann cell–neuron cocultures’, published in JCS. Clara is a PhD student in the lab of Peter Arthur-Farraj at the John Van Geest Centre for Brain Repair, University of Cambridge, UK, investigating axon–Schwann cell interactions after peripheral nervous system injury.

Influence of cold-stimulated adipocyte supernatant on the expression of adhesion-related molecules in Schwann cell line

Bell's palsy is the most common form of facial nerve palsy. This study aimed to explore the pathogenesis of Bell's palsy by investigating the effect of cold-stimulated adipocyte supernatant on adhesion molecule expression in Schwann cell line. Schwann cells were cultured in regular or adipocyte-conditioned medium and analyzed using RNA sequencing. The mRNA expression of Schwann cell adhesion molecules melanoma cell adhesion molecule (MCAM), protocadherin 9 (PCDH9), and intercellular cell adhesion molecule 1 (ICAM1) was determined using real-time reverse-transcription polymerase chain reaction. Differentially expressed genes were identified, and Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analyses were conducted. Compared with Schwann cells in 37 °C, the expression of MCAM, PCDH9, and ICAM1 was downregulated in Schwann cells treated with cold-stimulated adipocyte supernatant compared with Schwann cells in 37 °C. Adipocytes subjected to cold exposure may weaken the adhesion capacity of Schwann cells and disrupt the local homeostasis of Schwann cell–axon interactions by affecting the expression of MCAM, PCDH9, and ICAM1, ultimately leading to the development of demyelinating lesions.

In Vitro Myelination of Peripheral Axons in a Coculture of Rat Dorsal Root Ganglion Explants and Schwann Cells.

The process of myelination is essential to enable rapid and sufficient signal transduction in the nervous system. In the peripheral nervous system, neurons and Schwann cells engage in a complex interaction to control the myelination of axons. Disturbances of this interaction and breakdown of the myelin sheath are hallmarks of inflammatory neuropathies and occur secondarily in neurodegenerative disorders. Here, we present a coculture model of dorsal root ganglion explants and Schwann cells, which develops a robust myelination of peripheral axons to investigate the process of myelination in the peripheral nervous system, study axon-Schwann cell interactions, and evaluate the potential effects of therapeutic agents on each cell type separately. Methodologically, dorsal root ganglions of embryonic rats (E13.5) were harvested, dissociated from their surrounding tissue, and cultured as whole explants for 3 days. Schwann cells were isolated from 3-week-old adult rats, and sciatic nerves were enzymatically digested. The resulting Schwann cells were purified by magnetic-activated cell sorting and cultured under neuregulin and forskolin-enriched conditions. After 3 days of dorsal root ganglion explant culture, 30,000 Schwann cells were added to one dorsal root ganglion explant in a medium containing ascorbic acid. The first signs of myelination were detected on day 10 of coculture, through scattered signals for myelin basic protein in immunocytochemical staining. From day 14 onward, myelin sheaths were formed and propagated along the axons. Myelination can be quantified by myelin basic protein staining as a ratio of the myelination area and axon area, to account for the differences in axonal density. This model provides experimental opportunities to study various aspects of peripheral myelination in vitro, which is crucial for understanding the pathology of and possible treatment opportunities for demyelination and neurodegeneration in inflammatory and neurodegenerative diseases of the peripheral nervous system.

Schwann Cell and the Pathogenesis of Charcot-Marie-Tooth Disease.

Charcot-Marie-Tooth (CMT) disease is the most common hereditary neuropathy and genetically heterogeneous. CMT1 and CMTX are autosomal dominant and X-linked demyelinating neuropathies, respectively. CMT1A, CMT1B, and CMTX1 are the common forms of CMT, which are attributed to the genes encoding the myelin or gap junction proteins expressed in the myelinating Schwann cells. CMT4 is a rare autosomal recessive demyelinating neuropathy that usually shows an early-onset severe phenotype. Twelve genes have been described as CMT4, which encodes many kinds of proteins including mitochondrial proteins, phosphatases in the endosomal pathway, endocytic recycling proteins, and trafficking proteins. The genes responsible for CMT4 are expressed in Schwan cells and necessary for the development and maintenance in the peripheral nervous system. However, CMT1, CMT4, and CMTX1 are primarily demyelinating neuropathies, axonal degeneration is necessary for symptoms to develop. Schwann cell-axon interactions are impaired in the pathogenesis of demyelinating CMT.

Necl-4/Cadm4 recruits Par-3 to the Schwann cell adaxonal membrane.

Interactions between axons and Schwann cells are essential for the acquisition of Schwann cell radial and longitudinal polarity and myelin sheath assembly. In the internode, the largest of these longitudinal domains, axon-Schwann cell interactions are mediated by the Nectin-like (Necl) cell adhesion proteins, also known as SynCAMs or Cadms. In particular, Necl-1/Cadm3 expressed on the axon surface binds to Necl-4/Cadm4 expressed along the adaxonal membrane of myelinating Schwann cells. Necl-4 promotes myelination in vitro and is required for the timely onset of myelination and the fidelity of the organization of the myelin sheath and the internode in vivo. A key question is the identity of the downstream effectors of Necl-4 that mediate its effects. The cytoplasmic terminal region (CTR) of Necl-4 contains a PDZ-domain binding motif. Accordingly, we used the CTR of Necl-4 in an unbiased proteomic screen of PDZ-domain proteins. We identify Par-3, a multi-PDZ domain containing protein of the Par-aPKC polarity complex previously implicated in myelination, as an interacting protein. Necl-4 and Par-3 are colocalized along the inner Schwann cell membrane and coprecipitate from Schwann cell lysates. The CTR of Necl-4 binds to the first PDZ domain of Par-3 thereby recruiting Par-3 to sites of Necl-4/Necl-1 interaction. Knockdown of Necl-4 perturbs Par-3 localization to the inner membrane of Schwann cells in myelinating co-cultures. These findings implicate interactions of Necl-1/Necl-4 in the recruitment of Par-3 to the Schwann cell adaxonal membrane and the establishment of Schwann cell radial polarity.

3-2-01. Decreased motor axonal potassium currents in peripheral nerve hyperexcitability syndrome with negativity for anti-VGKC antibody and positivity for anti-CRMP5 antibody

Peripheral nerve hyperexcitability (PNH) syndromes are caused by spontaneous discharges originating from motor axons. Antibodies against voltage-gated potassium channels (VGKC) are detected in some patients with PNH syndrome, but the cause in the remaining patients has not yet been clarified. The index patient was a 46-year-old female with recurred thymoma and myasthenia gravis. Six month after the start of chemotherapy, she developed myokymia, fasciculations and muscle cramps in both her lower limbs. A nerve conduction studies revealed mild axonal polyneuropathy and a motor F-wave examination showed marked after-discharges. Needle electromyography showed fasciculation potentials and myokymic discharges. From these results, the patient was diagnosed with PNH syndrome. Nerve excitability study showed extremely greater supernormality in the recovery cycle and greater changes in depolarizing threshold electrotonus than control, suggesting decreased slow potassium currents in the motor axons. Serological tests revealed the absence of anti-VGKC antibody and the presence of anti-collapsin response mediator protein 5 (CRMP5) antibody. A recent study reported that CRMP5 plays an important role in axon–Schwann cell cooperation during development and nerve regeneration. The coexistence of nerve regeneration and anti-CRMP5 antibody may cause abnormal axon–Schwann cell interaction, which would result in slow potassium channel dysfunction and associated symptoms such as myokymia.

Betacellulin regulates schwann cell proliferation and myelin formation in the injured mouse peripheral nerve.

When a nerve fiber is cut or crushed, the axon segment that is separated from the soma degenerates distal from the injury in a process termed Wallerian degeneration (WD). C57BL/6OlaHsd-WldS (WldS ) mutant mice exhibit significant delays in WD. This results in considerably delayed Schwann cell and macrophage responses and thus in impaired nerve regenerations. In our previous work, thousands of genes were screened by DNA microarrays and over 700 transcripts were found to be differentially expressed in the injured sciatic nerve of WldS compared with wild-type (WT) mice. One of these transcripts, betacellulin (Btc), was selected for further analysis since it has yet to be characterized in the nervous system, despite being known as a ligand of the ErbB receptor family. We show that Btc mRNA is strongly upregulated in immature and dedifferentiated Sox2+ Schwann cells located in the sciatic nerve distal stump of WT mice, but not WldS mutants. Transgenic mice ubiquitously overexpressing Btc (Tg-Btc) have increased numbers of Schmidt-Lantermann incisures compared with WT mice, as revealed by Coherent anti-Stokes Raman scattering (CARS). Tg-Btc mice also have faster nerve conduction velocity. Finally, we found that deficiency in Btc reduces the proliferation of myelinating Schwann cells after sciatic nerve injury, while Btc overexpression induces Schwann cell proliferation and improves recovery of locomotor function. Taken together, these results suggest a novel regulatory role of Btc in axon-Schwann cell interactions involved in myelin formation and nerve repair. GLIA 2017 GLIA 2017;65:657-669.

A RET-ER81-NRG1 Signaling Pathway Drives the Development of Pacinian Corpuscles.

Communication between neurons and Schwann cells is critical for development, normal function, and regeneration of the peripheral nervous system. Despite many studies about axonal communication with myelinating Schwann cells, mostly via a specific isoform of Neuregulin1, the molecular nature of axonal communication with nonmyelinating Schwann cells is poorly understood. Here, we described a RET-ER81-Neuregulin1 signaling pathway in neurons innervating Pacinian corpuscle somatosensory end organs, which is essential for communication between the innervating axon and the end organ nonmyelinating Schwann cells. We also showed that this signaling pathway uses isoforms of Neuregulin1 that are not involved in myelination, providing evidence that neurons use different isoforms of Neuregulin1 to interact with different types of Schwann cells.

Intra-cardiac autonomic neuropathy in Holstein-Friesian dairy cows affected with bovine dilated cardiomyopathy

Three Holstein-Friesian dairy cows, aged 5 1/3 to 12 years, showed a history of heart failure and were euthanized on separate days. Commonly observed postmortem findings included edema in many sites of the body, dilatation and hypertrophy of the right cardiac ventricle, hepatic congestion, and swollen pale kidneys. Histopathologically, myocardial lesions were comprised of a variety of degenerative changes in cardiomyocytes and interstitial fibrosis, consistent with those described previously in cases of bovine dilated cardiomyopathy in Holstein-Friesian cattle. Additionally, in one of these three cows, unmyelinated nerve fibers of many intra-cardiac nerve bundles that probably were postganglionic fibers exhibited multifocal segmental degeneration of axons. Degenerated axons were closely associated with Schwann cell pathology, indicating an intimate involvement of axon-Schwann cell interaction. Such an intra-cardiac autonomic neuropathy might have had an adverse effect on the regulation of heart activity. It is not possible to determine if this intra-cardiac autonomic neuropathy was primary or secondary to the dilated cardiomyopathy.

Visualizing peripheral nerve regeneration by whole mount staining.

Peripheral nerve trauma triggers a well characterised sequence of events both proximal and distal to the site of injury. Axons distal to the injury degenerate, Schwann cells convert to a repair supportive phenotype and macrophages enter the nerve to clear myelin and axonal debris. Following these events, axons must regrow through the distal part of the nerve, re-innervate and finally are re-myelinated by Schwann cells. For nerve crush injuries (axonotmesis), in which the integrity of the nerve is maintained, repair may be relatively effective whereas for nerve transection (neurotmesis) repair will likely be very poor as few axons may be able to cross between the two parts of the severed nerve, across the newly generated nerve bridge, to enter the distal stump and regenerate. Analysing axon growth and the cell-cell interactions that occur following both nerve crush and cut injuries has largely been carried out by staining sections of nerve tissue, but this has the obvious disadvantage that it is not possible to follow the paths of regenerating axons in three dimensions within the nerve trunk or nerve bridge. To try and solve this problem, we describe the development and use of a novel whole mount staining protocol that allows the analysis of axonal regeneration, Schwann cell-axon interaction and re-vascularisation of the repairing nerve following nerve cut and crush injuries.

Axon-Schwann cell interactions during peripheral nerve regeneration in zebrafish larvae.

BackgroundPeripheral nerve injuries can severely affect the way that animals perceive signals from the surrounding environment. While damage to peripheral axons generally has a better outcome than injuries to central nervous system axons, it is currently unknown how neurons re-establish their target innervations to recover function after injury, and how accessory cells contribute to this task. Here we use a simple technique to create reproducible and localized injury in the posterior lateral line (pLL) nerve of zebrafish and follow the fate of both neurons and Schwann cells.ResultsUsing pLL single axon labeling by transient transgene expression, as well as transplantation of glial precursor cells in zebrafish larvae, we individualize different components in this system and characterize their cellular behaviors during the regenerative process. Neurectomy is followed by loss of Schwann cell differentiation markers that is reverted after nerve regrowth. We show that reinnervation of lateral line hair cells in neuromasts during pLL nerve regeneration is a highly dynamic process with promiscuous yet non-random target recognition. Furthermore, Schwann cells are required for directional extension and fasciculation of the regenerating nerve. We provide evidence that these cells and regrowing axons are mutually dependant during early stages of nerve regeneration in the pLL. The role of ErbB signaling in this context is also explored.ConclusionThe accessibility of the pLL nerve and the availability of transgenic lines that label this structure and their synaptic targets provides an outstanding in vivo model to study the different events associated with axonal extension, target reinnervation, and the complex cellular interactions between glial cells and injured axons during nerve regeneration.

Phosphorylation of LKB1/Par-4 establishes Schwann cell polarity to initiate and control myelin extent

The Schwann cell (SC)-axon interface represents a membrane specialization that integrates axonal signals to coordinate cytoskeletal dynamics resulting in myelination. Here we show that LKB1/Par-4 is asymmetrically localized to the SC-axon interface and colocalizes with the polarity protein Par-3. Using purified SCs and myelinating cocultures, we demonstrate that localization is dependent on the phosphorylation of LKB1 at serine-431. SC-specific deletion of LKB1 significantly attenuates developmental myelination, delaying the initiation and altering the myelin extent into adulthood, resulting in a 30% reduction in the conduction velocity along adult sciatic nerves. Phosphorylation of LKB1 by protein kinase A is essential to establish the asymmetric localization of LKB1 and Par-3 and rescues the delay in myelination observed in the SC-specific knockout of LKB1. Our findings suggest that SC polarity may coordinate multiple signaling complexes that couple SC-axon contact to the redistribution of specific membrane components necessary to initiate and control myelin extent.

Spinal cord organotypic slice cultures for the study of regenerating motor axon interactions with 3D scaffolds

Numerous in-vitro techniques exist for investigating the influence of 3D substrate topography on sensory axon growth. However, simple and cost-effective methods for studying post-natal motor axon interactions with such substrates are lacking. Here, spinal cord organotypic slice cultures (OSC) from post-natal day 7–9 rat pups were presented with spinal nerve roots, or blocks of fibrin hydrogel or 3D microporous collagen scaffolds to investigate motor axon–substrate interactions. By 7–14 days, axons from motor neuronal pools extended into the explanted nerve roots, growing along Schwann cell processes and demonstrating a full range of axon-Schwann cell interactions, from simple ensheathment to concentric wrapping by Schwann cell processes and the formation of compact myelin within a basal lamina sheath. Extensive motor axon regeneration and all stages of axon-Schwann interactions were also supported within the longitudinally orientated microporous framework of the 3D collagen scaffold. In stark contrast, the simple fibrin hydrogel only supported axon growth and cell migration over its surface. The relative ease of demonstrating such motor axon regeneration through the microporous 3D framework by immunofluorescence, two-photon microscopy and transmission electron microscopy strongly supports the adoption of this technique for assaying the influence of substrate topography and functionalization in regenerative bioengineering.

Myocilin Mediates Myelination in the Peripheral Nervous System through ErbB2/3 Signaling

The glaucoma-associated gene, myocilin, is expressed in ocular and non-ocular tissues including the peripheral nervous system, but its functions in these tissues remain poorly understood. We demonstrate that in sciatic nerve, myocilin is expressed in Schwann cells with high concentrations at the nodes of Ranvier. There, myocilin interacts with gliomedin, neurofascin, and NrCAM, which are essential for node formation and function. Treatment of isolated dorsal root ganglion cultures with myocilin stimulates clustering of the nodal proteins neurofascin and sodium channel Nav1.2. Sciatic nerves of myocilin null mice express reduced levels of several myelin-associated and basal membrane proteins compared with those of wild-type littermates. They also demonstrate reduced myelin sheath thickness and partial disorganization of the nodes. Myocilin signaling through ErbB2/3 receptors may contribute to these observed effects. Myocilin binds to ErbB2/ErbB3, activates these receptors, and affects the downstream PI3K-AKT signaling pathway. These data implicate a role for myocilin in the development and/or maintenance of myelination and nodes of Ranvier in sciatic nerve.

Tetraspanins are involved in Schwann cell–axon interaction

Many studies have shown that tetraspanins play important role in cell-cell and cell-extracellular matrix (ECM) interactions. The repertoire and functions of tetraspanins in Schwann cells, glial cells of the peripheral nervous system have remained largely uncharacterized. This study was undertaken to identify Schwann cell tetraspanins and to elucidate their possible functions. Microarray analysis revealed the expression of numerous tetraspanins in primary culture of Schwann cells. Expression of five of them, CD9, CD63, CD81, CD82, and CD151, and of tetraspanin-associated protein EWI-2 was also confirmed by immunofluorescence. Localization of CD9, CD63, CD81, and EWI-2 was largely confined to paranodes and Schmidt-Lanterman incisures, regions of noncompact myelin. Immunoprecipitation experiments showed that these four proteins form a complex in Schwann cells. siRNA silencing of individual components of the complex did not affect Schwann cell adhesion to ECM proteins or attachment to and alignment with axons. However, suppression of both CD63 and CD81 expression together significantly inhibited extension of Schwann cell processes along axons, without affecting initial attachment of the cells to the axonal surface. Adhesion, spreading, and migration of Schwann cells on ECM proteins also were not affected by double silencing of CD63 and CD81. Suppression of CD63 and CD81 expression did not affect the ability of Schwann cells to myelinate dorsal root ganglion neurons in vitro. These findings strongly suggest that CD63 and CD81 play an important role in Schwann cell spreading along axons but seem to be dispensable for Schwann cell myelination.

Reduction of the ST6 β-Galactosamide α-2,6-Sialyltransferase 1 (ST6GAL1)-catalyzed Sialylation of Nectin-like Molecule 2/Cell Adhesion Molecule 1 and Enhancement of ErbB2/ErbB3 Signaling by MicroRNA-199a

Nectin-like molecule 2 (Necl-2)/cell adhesion molecule 1 (CADM1) is shown to be down-regulated by the promoter hypermethylation and/or loss of heterozygosity at chromosome 11q23.2 in many types of cancers, including lung and breast cancers, and is proposed to serve as a tumor suppressor. However, the incidence of these epigenetic and genetic abnormalities of Necl-2 is 30-60% in these cancers, and other mechanisms for the suppression of Necl-2 are presumed to be present. We previously showed that Necl-2 interacts in cis with ErbB3 and suppresses the heregulin (HRG)-induced ErbB2/ErbB3 signaling for cell movement and death. We studied here the relationship between Necl-2 and microRNA-199a (miR-199a) that is up-regulated or down-regulated in a variety of cancers. miR-199a did not directly target the Necl-2 mRNA or affect its mRNA level in human lung cancer A549 cells and human embryonic kidney HEK293 cells. Necl-2 was at least sialylated by the sialyltransferase ST6 β-galactosamide α-2,6-sialyltransferase 1 (ST6GAL1). miR-199a targeted ST6GAL1 and reduced both the sialylation and the protein level of Necl-2. In addition, miR-199a enhanced the HRG-induced ErbB2/ErbB3 signaling. These results indicate that the suppressive role of Necl-2 in the HRG-induced ErbB2/ErbB3 signaling is regulated by miR-199a at least through the reduction of the ST6GAL1-catalyzed sialylation of Necl-2 and/or through the reduction of the protein level of Necl-2 presumably by the protein degradation.

Schwann Cell LRP1 Regulates Remak Bundle Ultrastructure and Axonal Interactions to Prevent Neuropathic Pain

Trophic support and myelination of axons by Schwann cells in the PNS are essential for normal nerve function. Herein, we show that deletion of the LDL receptor-related protein-1 (LRP1) gene in Schwann cells (scLRP1(-/-)) induces abnormalities in axon myelination and in ensheathment of axons by nonmyelinating Schwann cells in Remak bundles. These anatomical changes in the PNS were associated with mechanical allodynia, even in the absence of nerve injury. In response to crush injury, sciatic nerves in scLRP1(-/-) mice showed accelerated degeneration and Schwann cell death. Remyelinated axons were evident 20 d after crush injury in control mice, yet were largely absent in scLRP1(-/-) mice. In the partial nerve ligation model, scLRP1(-/-) mice demonstrated significantly increased and sustained mechanical allodynia and loss of motor function. Evidence for central sensitization in pain processing included increased p38MAPK activation and activation of microglia in the spinal cord. These studies identify LRP1 as an essential mediator of normal Schwann cell-axonal interactions and as a pivotal regulator of the Schwann cell response to PNS injury in vivo. Mice in which LRP1 is deficient in Schwann cells represent a model for studying how abnormalities in Schwann cell physiology may facilitate and sustain chronic pain.