The claw paw mutation reveals a role for Lgi4 in peripheral nerve development

Peripheral nerve development results from multiple cellular interactions between axons, Schwann cells and the surrounding mesenchymal tissue. The delayed axonal sorting and hypomyelination throughout the peripheral nervous system of claw paw (clp) mutant mice suggest that the clp gene product is critical for these interactions. Here we identify the clp mutation as a 225-bp insertion in the Lgi4 gene. Lgi4 encodes a secreted and glycosylated leucine-rich repeat protein and is expressed in Schwann cells. The clp mutation affects Lgi4 mRNA splicing, resulting in a mutant protein that is retained in the cell. Additionally, siRNA-mediated downregulation of Lgi4 in wild-type neuron-Schwann cell cocultures inhibits myelination, whereas exogenous Lgi4 restores myelination in clp/clp cultures. Thus, the abnormalities observed in clp mice are attributable to the loss of Lgi4 function, and they identify Lgi4 as a new component of Schwann cell signaling pathway(s) that controls axon segregation and myelin formation.     

N-Acetylated alpha-linked acidic dipeptidase may be involved in axon-Schwann cell signalling

Summary N-Acetylated alpha-linked acidic dipeptidase is a membrane-bound peptidase that cleaves the neuropeptide N-acetylaspartyl-glutamate to N-acetyl-aspartate and glutamate. Previously, we have shown that in adult rat this enzyme is expressed by the non-myelinating Schwann cells in peripheral nerve. In the present study, we have determined the expression pattern of this peptidase in rat sciatic nerve during late embryonal and early postnatal development, using double-label immunofluorescence, enzyme assays and immunoblotting. We demonstrate that N-acethylated alpha-linked acidic dipeptidase is expressed by all Schwann cell precursor cells on embryonal day 14/15 and by all undifferentiated Schwann cells on embryonal days 16/17 and 20/21 and postnatal day 1. Moreover, we show that during the first postnatal week, the peptidase expression is down-regulated in the myelinating Schwann cells while the total enzyme activity levels and the enzyme amounts present in the nerve are transiently increased. To determine whether Schwann cell peptidase expression is dependent on axonal contact, we performed immunofluorescence experiments in cultured Schwann cells. Thesein vitro experiments demonstrate that the expression of this enzyme is maintained in culture for several weeks without axonal contact. Furthermore, they confirm previous suggestions that this peptidase is expressed on the extracellular side of the Schwann cell membrane. These findings support the notion that N-acetylated alpha-linked acidic dipeptidase takes part in signaling between peripheral axons and Schwann cells. The temporary increase in peptidase activity during the first postnatal week strongly implicates a role for this enzyme in the process of axon ensheathment and/or axon myelination.     

Abnormal Schwann cell/axon interactions in the Trembler-J mouse

The Trembler-J ( Tr ^J ) mouse has a point mutation in the gene coding for peripheral myelin protein 22 (PMP22). Disturbances in PMP22 are associated with abnormal myelination in a range of inherited peripheral neuropathies both in mice and humans. PMP22 is produced mainly by Schwann cells in the peripheral nervous system where it is localised to compact myelin. The function of PMP22 is unclear but its low abundance (∼5% of total myelin protein) means that it is unlikely to play a structural role. Its inclusion in a recently discovered family of proteins suggests a function in cell proliferation/differentiation and possibly in adhesion. Nerves from Tr ^J and the allelic Trembler ( Tr ) mouse are characterised by abnormally thin myelin for the size of the axon and an increased number of Schwann cells. We report ultrastructural evidence of abnormal Schwann cell-axon interactions. Schwann cell nuclei have been found adjacent to the nodes of Ranvier whereas in normal animals they are located near the centre of the internodes. In some fibres the terminal myelin loops faced outwards into the extracellular space instead of turning inwards and terminating on the axon. In severely affected nerves many axons were only partially surrounded by Schwann cell cytoplasm. All these features suggest a failure of Schwann cell–axon recognition or interaction. In addition to abnormalities related to abnormal myelination there was significant axonal loss in the dorsal roots.     

Double myelination of axons in the sympathetic nervous system

Summary Relationships between axons and Schwann cells in myelinated fibres of the superior cervical (sympathetic) ganglion have been examined in normal adult rats. In cross-sections through the ganglion up to 4 % of myelinated fibres were focally encircled by an additional myelinating Schwann cell, forming regions termed double myelination. In these regions and elsewhere in the ganglion, the structure of the inner fibre (axon and myelinating Schwann cell) conformed to the relationships expected on the basis of numerous previous investigations on normal peripheral nerve. However, the outer Schwann cell and myelin sheath, which formed an annulus around the inner fibre, was remarkable in that it apparently made no direct contact either with the centrally enclosed axon or with any neighbouring axon, yet appeared largely if not completely intact. In addition, the increasing frequency of double myelination in older animals and the rarity of myelin degeneration in the same ganglia indicate that the outer Schwann cell, and in particular its myelin sheath, persist for some period in an isolated form. Double myelination was not located in non-sympathetic peripheral nerve samples from the same animals. Double myelination may result from the displacement of one myelin internode by the interposition of another Schwann cell rendering the original Schwann cell redundant. There was no involvement of haematogenous cells as occurs in some demyelinating conditions. While some parallels may be found with previous studies, this would appear to be the first report of apparent survival of myelin in a Schwann cell not making, as far as could be determined in the present study, at least partial direct axonal contact. These observations on sympathetic nerve may provide a new perspective on axon-Schwann cell signalling.     

A novel molecular interaction for the adhesion of follicular CD4 T cells to follicular DC

Nectins and Nectin‐like molecules (Necl) play a critical role in cell polarity within epithelia and in the nervous and reproductive systems. Recently, immune receptors specific for Nectins/Necl have been described. Since the expression and distribution of Nectins/Necl is often subverted during tumorigenesis, it has been suggested that the immune system may use these receptors to recognize and eliminate tumors. Here we describe a novel immunoreceptor, Washington University Cell Adhesion Molecule, which is expressed on human follicular B helper T cells (TFH) and binds a Nectin/Necl family member, the poliovirus receptor (PVR), under both static and flow conditions. Furthermore, we demonstrate that PVR is abundantly expressed by follicular DC (FDC) within the germinal center. These results reveal a novel molecular interaction that mediates adhesion of TFH to FDC and provide the first evidence that immune receptors for Nectins/Necl may be involved the generation of T cell‐dependent antibody responses.     

Pard3 regulates contact between neural crest cells and the timing of Schwann cell differentiation but is not essential for neural crest migration or myelination

Background : Schwann cells, which arise from the neural crest, are the myelinating glia of the peripheral nervous system. During development neural crest and their Schwann cell derivatives engage in a sequence of events that comprise delamination from the neuroepithelium, directed migration, axon ensheathment, and myelin membrane synthesis. At each step neural crest and Schwann cells are polarized, suggesting important roles for molecules that create cellular asymmetries. In this work we investigated the possibility that one polarity protein, Pard3, contributes to the polarized features of neural crest and Schwann cells that are associated with directed migration and myelination. Results : We analyzed mutant zebrafish embryos deficient for maternal and zygotic pard3 function. Time‐lapse imaging revealed that neural crest delamination was normal but that migrating cells were disorganized with substantial amounts of overlapping membrane. Nevertheless, neural crest cells migrated to appropriate peripheral targets. Schwann cells wrapped motor axons and, although myelin gene expression was delayed, myelination proceeded to completion. Conclusions : Pard3 mediates contact inhibition between neural crest cells and promotes timely myelin gene expression but is not essential for neural crest migration or myelination. Developmental Dynamics 243:1511–1523, 2014. © 2014 Wiley Periodicals, Inc.     

Node of Ranvier formation on motoneurons in vitro

One of the most significant interactions between Schwann cells and neurons is myelin sheath formation. Myelination is a vertebrate adaptation that enables rapid conduction of action potentials without a commensurate increase in axon diameter. In vitro neuronal systems provide a unique modality to study both factors influencing myelination and diseases associated with myelination. Currently, no in vitro system for motoneuron myelination by Schwann cells has been demonstrated. This work details the myelination of motoneuron axons by Schwann cells, with complete Node of Ranvier formation, in a defined in vitro culture system. This defined system utilizes a novel serum-free medium in combination with the non-biological substrate, N-1[3 (trimethoxysilyl) propyl] diethylenetriamine (DETA). The myelinated segments and nodal proteins were visualized and quantified using confocal microscopy. This defined system provides a highly controlled, reproducible model for studying Schwann cell interactions with motoneurons as well as the myelination process and its effect on neuronal plasticity. Furthermore, an in vitro system that would allow studies of motoneuron myelination would be beneficial for understanding peripheral demyelinating neuropathies such as diabetes induced peripheral neuropathy and could lead to a better understanding of CNS demyelinating diseases like multiple sclerosis, as well as neuromuscular junction maturation and maintenance.     

NF1 loss disrupts Schwann cell–axonal interactions: a novel role for semaphorin 4F

Neurofibromatosis type 1 (NF1) patients develop neurofibromas, tumors of Schwann cell origin, as a result of loss of the Ras-GAP neurofibromin. In normal nerves, Schwann cells are found tightly associated with axons, while loss of axonal contact is a frequent and important early event in neurofibroma development. However, the molecular basis of this physical interaction or how it is disrupted in cancer remains unclear. Here we show that loss of neurofibromin in Schwann cells is sufficient to disrupt Schwann cell/axonal interactions via up-regulation of the Ras/Raf/ERK signaling pathway. Importantly, we identify down-regulation of semaphorin 4F (Sema4F) as the molecular mechanism responsible for the Ras-mediated loss of interactions. In heterotypic cocultures, Sema4F knockdown induced Schwann cell proliferation by relieving axonal contact-inhibitory signals, providing a mechanism through which loss of axonal contact contributes to tumorigenesis. Importantly, Sema4F levels were strongly reduced in a panel of human neurofibromas, confirming the relevance of these findings to the human disease. This work identifies a novel role for the guidance-molecules semaphorins in the mediation of Schwann cell/axonal interactions, and provides a molecular mechanism by which heterotypic cell–cell contacts control cell proliferation and suppress tumorigenesis. Finally, it provides a new approach for the development of therapies for NF1.     

Expression and function of alpha 4/beta 7 integrin on human natural killer cells.

In this report we have analysed the expression and function of the alpha 4/beta 7 heterodimer in human natural killer (NK) cells. The expression of alpha 4 beta 7 is induced in NK cells upon activation as the anti alpha 4 beta 2 ACT-1 monoclonal antibody (mAb) family stained a minority of peripheral blood NK cells, whereas it strongly reacted with in vitro long-term interleukin-2 (IL-2)-activated NK cells. Incubation with ACT-1 on its F(ab) fragments induced a strong homotypic adhesion of NK cells, comparable to than stimulated by the anti-alpha 4 HPI 7 mAb. Cell cell interaction induced by the ACT-1 mAb was only prevented by another anti-alpha 4 mAb (HP2.1) that recognizes a different epitope. In alpha 4 beta 7-mediated cell aggregation the alpha 4 beta 7 heterodimer was redistributed to intercellular contact sites thus, suggesting a direct involvement of this integrin in the formation of cell clusters. In NK cells attached to Fibronectin (FN38) or vascular cell adhesion molecule-1 (VCAM-1), both alpha 4 beta 7 and alpha 4 beta 7 integrins were redistributed at the ventral cellular membrane forming discrete contact sites. The ACT-1 mAb only partially blocked NK cell binding to FN38, but in combination with the anti-beta 7 mAb LIAI 2, NK cell binding to FN38 was completely inhibited. In contrast. ACT-1 did not modify NK cell adhesion to VCAM-1 thus supporting the theory that the alpha 4 beta 7 binding sites for both ligands appear to be different. Our results indicate that upon IL-2-activation, expression of functional alpha 4 beta-integrin is induced on NK cells potentially participating in their interaction with both extracellular matrix and endothelial cells.     

Interaction of Necl‐4/CADM4 with ErbB3 and integrin α6β4 and inhibition of ErbB2/ErbB3 signaling and hemidesmosome disassembly

Nectin‐like molecule 4 (Necl‐4)/CADM4, a transmembrane cell–cell adhesion molecule with three Ig‐like domains, was shown to serve as a tumor suppressor, but its mode of action has not been elucidated. In this study, we showed that Necl‐4 interacted in cis with ErbB3 through their extracellular regions, recruited PTPN13 and inhibited the heregulin‐induced activation of the ErbB2/ErbB3 signaling. In addition, we extended our previous finding that Necl‐4 interacts in cis with integrin α₆β₄ through their extracellular regions and found that Necl‐4 inhibited the phorbol ester‐induced disassembly of hemidesmosomes. These results indicate that Necl‐4 serves as a tumor suppressor by inhibiting the ErbB2/ErbB3 signaling and hemidesmosome disassembly.     

Development of early postnatal peripheral nerve abnormalities in Trembler-J and PMP22 transgenic mice

Mutations in the gene for peripheral myelin protein 22 ( PMP22 ) are associated with peripheral neuropathy in mice and humans. Although PMP22 is strongly expressed in peripheral nerves and is localised largely to the myelin sheath, a dual role has been suggested as 2 differentially expressed promoters have been found. In this study we compared the initial stages of postnatal development in transgenic mouse models which have, in addition to the murine pmp22 gene, 7 (C22) and 4 (C61) copies of the human PMP22 gene and in homozygous and heterozygous Trembler-J ( Tr ^J ) mice, which have a point mutation in the pmp22 gene. The number of axons that were singly ensheathed by Schwann cells was the same in all groups indicating that PMP22 does not function in the initial ensheathment and separation of axons. At both P4 and P12 all mutants had an increased proportion of fibres that were incompletely surrounded by Schwann cell cytoplasm indicating that this step is disrupted in PMP22 mutants. C22 and homozygous Tr ^J animals could be distinguished by differences in the Schwann cell morphology at the initiation of myelination. In homozygous Tr ^J animals the Schwann cell cytoplasm had failed to make a full turn around the axon whereas in the C22 strain most fibres had formed a mesaxon. It is concluded that PMP22 functions in the initiation of myelination and probably involves the ensheathment of the axon by the Schwann cell, and the extension of this cell along the axon. Abnormalities may result from a failure of differentiation but more probably from defective interactions between the axon and the Schwann cell.     

Developmental expression of P0 mRNA and P0 protein in the sciatic nerve and the spinal nerve roots of the rat

Summary Expression of myelin P0 protein by myelinating Schwann cellsin vivo is dependent on axonal influences. This report describes P0 gene expression during development of rat sciatic nerve and spinal nerve roots using Northern blotting,in situ hybridization and immunohistochemistry. We demonstrate that: (1) the appearance of P0 mRNA and P0 protein in Schwann cells during nerve development in the rat begins prenatally, at day 18 post-fertilization (E18); (2) P0 mRNA and P0 protein have essentially identical developmental profiles, and are expressed in Schwann cells that are many days prior to myelin formation; (3) initial P0 gene expression is greatest in Schwann cells at the periphery of nerve bundles and in Schwann cells in contact with motor axons; (4) the decline in P0 expression with nerve maturation is accompanied by a sharp decline in P0 message levels in most Schwann cells, but a small subpopulation of these cells continue to synthesize very high levels of P0 mRNA. This study provides data on myelin P0 protein gene expression and distribution during PNS development and adds further insights into the axonal influences controlling Schwann cell behaviour during myelination of the rat PNS.     

Double myelination of axons in the sympathetic nervous system of the mouse. II. Mechanisms of formation

Summary The phenomenon termed double myelination, present in sympathetic nerve of normal adult rats and mice, comprises regions of a myelinated axon which are concentrically ensheathed by additional (outer) myelinating Schwann cells. Evidence has been presented that in some instances the outer Schwann cell fails to make contact with an axon, yet its myelin sheath characteristically remains ultrastructurally intact. The present study has sought to identify and analyse configurations intermediate between single and double myelination, in order to determine the mechanism(s) underlying the formation of double ensheathment. Superior cervical ganglia from normal male mice aged 12–24 months were prepared for electron microscopy by systemic aldehyde perfusion. Regions of interest were extensively serial-sectioned for detailed electron microscopical analysis and reconstruction. The earliest evidence for alteration to the expected intimate ensheathment of axons by myelinating Schwann cells involved invasion of supernumerary Schwann cells and their processes at the node of Ranvier, resulting in displacement of the paranodal pockets from axonal contact. Similar paranodal displacement occurred at heminodes as a result of lateral extension and invasion of processes from the adjacent Schwann cell (i.e. the cell investing the unmyelinated domain of the axon). Subsequently, processes of the invading cell extended progressively into internodal regions, located at all times between the plasma membranes of the axon and displaced Schwann cell. The cytoplasmic pockets at the remaining paranode were then subject to invasion. At various stages of displacement myelin formation commenced within the invading cell, representing the first acquisition of double myelin ensheathment in the development of the configuration. Involvement of haematogenous cells in displacement was not detected. There was also evidence consistent with paranodal displacement by adjacent pre-existing myelinating cells, but this additional mechanism appeared minor relative to the involvement of (initially) non-myelinating Schwann cells. We found no evidence for the alternative possibility that Schwann cells could synthesize a myelin sheath around a pre-existing myelinated axonde novo, independent of any direct axonal contact. These results are consistent with the well-established requirement for axonal contact by Schwann cells engaging in initial myelin formation, in the sense that the myelin sheath of the outer cell was synthesized prior to its displacement, and that a myelin sheath was not formed by the invading cell until it had invested the axon in a 1:1 relationship. In addition, the emergence of several key features of double myelination (infolding and continuing integrity of the outer sheath, sites of presence/absence of basal lamina on the outer cell) further supports the view that double myelination represents a culmination of these developmental stages.