Patrice Maurel

Nectin-like proteins mediate axon–Schwann cell interactions along the internode and are essential for myelination

Axon–glial interactions are critical for the induction of myelination and the domain organization of myelinated fibers. Although molecular complexes that mediate these interactions in the nodal region are known, their counterparts along the internode are poorly defined. We report that neurons and Schwann cells express distinct sets of nectin-like (Necl) proteins: axons highly express Necl-1 and -2, whereas Schwann cells express Necl-4 and lower amounts of Necl-2. These proteins are strikingly localized to the internode, where Necl-1 and -2 on the axon are directly apposed by Necl-4 on the Schwann cell; all three proteins are also enriched at Schmidt-Lanterman incisures. Binding experiments demonstrate that the Necl proteins preferentially mediate heterophilic rather than homophilic interactions. In particular, Necl-1 on axons binds specifically to Necl-4 on Schwann cells. Knockdown of Necl-4 by short hairpin RNA inhibits Schwann cell differentiation and subsequent myelination in cocultures. These results demonstrate a key role for Necl-4 in initiating peripheral nervous system myelination and implicate the Necl proteins as mediators of axo–glial interactions along the internode.

Chondroitin sulfate proteoglycans in the developing central nervous system. I. Cellular sites of synthesis of neurocan and phosphacan

We have used in situ hybridization histochemistry to examine the cellular sites of synthesis of two major nervous tissue proteoglycans, neurocan and phosphacan, in embryonic and postnatal rat brain and spinal cord. Both proteoglycans were detected only in nervous tissue. Neurocan mRNA was evident in neurons, including cerebellar granule cells and Purkinje cells, and in neurons of the hippocampal formation and cerebellar nuclei. In contrast, phosphacan message was detected only in astroglia, such as the Golgi epithelial cells of the cerebellum. At embryonic day 13–16, phosphacan mRNA is largely confined to areas of active cell proliferation (e.g., the ventricular zone of the ganglionic eminence and septal area of the brain and the ependymal layer surrounding the central canal of the spinal cord) as well as being present in the roof plate. The distribution of neurocan message is more widespread, extending to the cortex, hippocampal formation, caudate putamen, and basal telencephalic neuroepithelium, and neurocan mRNA is present in both the ependymal and mantle layers of the spinal cord but not in the roof plate. The presence of neurocan mRNA in areas where the proteoglycan is not expressed suggests that the short open reading frame in the 5′‐leader of neurocan may function as a cis‐acting regulatory signal for the modulation of neurocan expression in the developing central nervous system.

Soluble Neuregulin-1 Has Bifunctional, Concentration-Dependent Effects on Schwann Cell Myelination

Members of the neuregulin-1 (Nrg1) growth factor family play important roles during Schwann cell development. Recently, it has been shown that the membrane-bound type III isoform is required for Schwann cell myelination. Interestingly, however, Nrg1 type II, a soluble isoform, inhibits the process. The mechanisms underlying these isoform-specific effects are unknown. It is possible that myelination requires juxtacrine Nrg1 signaling provided by the membrane-bound isoform, whereas paracrine stimulation by soluble Nrg1 inhibits the process. To investigate this, we asked whether Nrg1 type III provided in a paracrine manner would promote or inhibit myelination. We found that soluble Nrg1 type III enhanced myelination in Schwann cell-neuron cocultures. It improved myelination of Nrg1 type III+/− neurons and induced myelination on normally nonmyelinated sympathetic neurons. However, soluble Nrg1 type III failed to induce myelination on Nrg1 type III−/− neurons. To our surprise, low concentrations of Nrg1 type II also elicited a similar promyelinating effect. At high doses, however, both type II and III isoforms inhibited myelination and increased c-Jun expression in a manner dependent on Mek/Erk (mitogen-activated protein kinase kinase/extracellular signal-regulated kinase) activation. These results indicate that paracrine Nrg1 signaling provides concentration-dependent bifunctional effects on Schwann cell myelination. Furthermore, our studies suggest that there may be two distinct steps in Schwann cell myelination: an initial phase dependent on juxtacrine Nrg1 signaling and a later phase that can be promoted by paracrine stimulation.

p38 MAPK Activation Promotes Denervated Schwann Cell Phenotype and Functions as a Negative Regulator of Schwann Cell Differentiation and Myelination

Physical damage to the peripheral nerves triggers Schwann cell injury response in the distal nerves in an event termed Wallerian degeneration: the Schwann cells degrade their myelin sheaths and dedifferentiate, reverting to a phenotype that supports axon regeneration and nerve repair. The molecular mechanisms regulating Schwann cell plasticity in the PNS remain to be elucidated. Using both in vivo and in vitro models for peripheral nerve injury, here we show that inhibition of p38 mitogen-activated protein kinase (MAPK) activity in mice blocks Schwann cell demyelination and dedifferentiation following nerve injury, suggesting that the kinase mediates the injury signal that triggers distal Schwann cell injury response. In myelinating cocultures, p38 MAPK also mediates myelin breakdown induced by Schwann cell growth factors, such as neuregulin and FGF-2. Furthermore, ectopic activation of p38 MAPK is sufficient to induce myelin breakdown and drives differentiated Schwann cells to acquire phenotypic features of immature Schwann cells. We also show that p38 MAPK concomitantly functions as a negative regulator of Schwann cell differentiation: enforced p38 MAPK activation blocks cAMP-induced expression of Krox 20 and myelin proteins, but induces expression of c-Jun. As expected of its role as a negative signal for myelination, inhibition of p38 MAPK in cocultures promotes myelin formation by increasing the number as well as the length of individual myelin segments. Altogether, our data identify p38 MAPK as an important regulator of Schwann cell plasticity and differentiation.

Localization of aggrecan and versican in the developing rat central nervous system

The localization of aggrecan and mRNA splice variants of versican in the developing rat central nervous system has been examined by using specific polyclonal antibodies to the nonhomologous glycosaminoglycan attachment regions of these hyaluronan‐binding chondroitin sulfate proteoglycans. At embryonic day 16 (E16), aggrecan and versican splice variants containing either or both the α‐and β‐domains are present in the marginal zone and subplate of the cerebral cortex and in the amygdala, internal capsule, and the optic and lateral olfactory tracts. There is strong staining of versican but not of aggrecan in the hippocampus and dentate gyrus by E19, whereas both aggrecan and α‐versican are present in the fimbria. At E19, aggrecan is seen throughout the cerebral cortex, whereas the distribution of versican is considerably more limited, being confined essentially to the marginal zone and subplate. At 1 week postnatal, both aggrecan and versican are present in the prospective white matter and in the molecular and granule cell layers of the cerebellum, but neither proteoglycan is seen in the external granule cell layer. α‐ but not β‐versican staining is seen in Purkinje cells, and aggrecan staining of Purkinje cells is also rather minimal. In the spinal cord at E13, aggrecan is present in the dorsal root entry zone, ventral funiculus, mantle layer, and floor plate, as well as in the dorsal root ganglia and ventral roots. However, α‐versican is confined to the dorsal root entry zone and the ependyma surrounding the spinal canal, and β‐versican is not present in spinal cord parenchyma at this developmental stage, being limited to the surrounding connective tissue. By E19, there are significant amounts of all three proteoglycans in the spinal cord. Aggrecan staining is most intense in the lateral funiculus and the fasciculi gracilis and cuneatus, where α‐versican staining is also strong. In contrast, β‐versican is seen predominantly in the motor columns. Differences in the localization and temporal expression patterns of these chondroitin sulfate proteoglycans suggest that, like neurocan and phosphacan, they have partially complementary roles during central nervous system development. Development Dynamics 227:143–149, 2003.

Differential Expression of Proteoglycans at Central and Peripheral Nodes of Ranvier

The nodes of Ranvier are regularly spaced gaps between myelin sheaths that are markedly enriched in voltage‐gated sodium channels and associated proteins. Myelinating glia play a key role in promoting node formation, although the requisite glial signals remain poorly understood. In this study, we have examined the expression of glial proteoglycans in the peripheral and central nodes. We report that the heparan sulfate proteoglycan, syndecan‐3, becomes highly enriched with PNS node formation; its ligand, collagen V, is also concentrated at the PNS nodes and at lower levels along the abaxonal membrane. The V1 isoform of versican, a chondroitin sulfate proteoglycan, is also present in the nodal gap. By contrast, CNS nodes are enriched in versican isoform V2, but not syndecan‐3. We have examined the molecular composition of the PNS nodes in syndecan‐3 knockout mice. Nodal components are normally expressed in mice deficient in syndecan‐3, suggesting that it has a nonessential role in the organization of nodes in the adult. These results indicate that the molecular composition and extracellular environment of the PNS and CNS nodes of Ranvier are significantly distinct.

The 4.1B Cytoskeletal Protein Regulates the Domain Organization and Sheath Thickness of Myelinated Axons

Myelinated axons are organized into specialized domains critical to their function in saltatory conduction, i.e., nodes, paranodes, juxtaparanodes, and internodes. Here, we describe the distribution and role of the 4.1B protein in this organization. 4.1B is expressed by neurons, and at lower levels by Schwann cells, which also robustly express 4.1G. Immunofluorescence and immuno‐EM demonstrates 4.1B is expressed subjacent to the axon membrane in all domains except the nodes. Mice deficient in 4.1B have preserved paranodes, based on marker staining and EM in contrast to the juxtaparanodes, which are substantially affected in both the PNS and CNS. The juxtaparanodal defect is evident in developing and adult nerves and is neuron‐autonomous based on myelinating cocultures in which wt Schwann cells were grown with 4.1B‐deficient neurons. Despite the juxtaparanodal defect, nerve conduction velocity is unaffected. Preservation of paranodal markers in 4.1B deficient mice is associated with, but not dependent on an increase of 4.1R at the axonal paranodes. Loss of 4.1B in the axon is also associated with reduced levels of the internodal proteins, Necl‐1 and Necl‐2, and of alpha‐2 spectrin. Mutant nerves are modestly hypermyelinated and have increased numbers of Schmidt‐Lanterman incisures, increased expression of 4.1G, and express a residual, truncated isoform of 4.1B. These results demonstrate that 4.1B is a key cytoskeletal scaffold for axonal adhesion molecules expressed in the juxtaparanodal and internodal domains that unexpectedly regulates myelin sheath thickness.

E-cadherin enhances neuregulin signaling and promotes Schwann cell myelination.

In myelinating Schwann cells, E‐cadherin is a component of the adherens junctions that stabilize the architecture of the noncompact myelin region. In other cell types, E‐cadherin has been considered as a signaling receptor that modulates intracellular signal transduction and cellular responses. To determine whether E‐cadherin plays a regulatory role during Schwann cell myelination, we investigated the effects of E‐cadherin deletion and over‐expression in Schwann cells. In vivo, Schwann cell‐specific E‐cadherin ablation results in an early myelination delay. In Schwann cell‐dorsal root ganglia neuron co‐cultures, E‐cadherin deletion attenuates myelin formation and shortens the myelin segment length. When over‐expressed in Schwann cells, E‐cadherin improves myelination on Nrg1 type III+/− neurons and induces myelination on normally non‐myelinated axons of sympathetic neurons. The pro‐myelinating effect of E‐cadherin is associated with an enhanced Nrg1‐erbB receptor signaling, including activation of the downstream Akt and Rac. Accordingly, in the absence of E‐cadherin, Nrg1‐signaling is diminished in Schwann cells. Our data also show that E‐cadherin expression in Schwann cell is induced by axonal Nrg1 type III, indicating a reciprocal interaction between E‐cadherin and the Nrg1 signaling. Altogether, our data suggest a regulatory function of E‐cadherin that modulates Nrg1 signaling and promotes Schwann cell myelin formation. GLIA 2015;63:1522–1536

Nectin-like 4 complexes with Choline Transporter-Like protein-1, and regulates Schwann cell choline homeostasis and lipid biogenesis in vitro

Nectin-like 4 (NECL4, CADM4) is a Schwann cell-specific cell adhesion molecule that promotes axo-glial interactions. In vitro and in vivo studies have shown that NECL4 is necessary for proper peripheral nerve myelination. However, the molecular mechanisms that are regulated by NECL4 and affect peripheral myelination currently remain unclear. We used an in vitro approach to begin identifying some of the mechanisms that could explain NECL4 function. Using mass spectrometry and Western blotting techniques, we have identified choline transporter-like 1 (CTL1) as a putative complexing partner with NECL4. We show that intracellular choline levels are significantly elevated in NECL4-deficient Schwann cells. The analysis of extracellular d9-choline uptake revealed a deficit in the amount of d9-choline found inside NECL4-deficient Schwann cells, suggestive of either reduced transport capabilities or increased metabolization of transported choline. An extensive lipidomic screen of choline derivatives showed that total phosphatidylcholine and phosphatidylinositol (but not diacylglycerol or sphingomyelin) are significantly elevated in NECL4-deficient Schwann cells, particularly specific subspecies of phosphatidylcholine carrying very long polyunsaturated fatty acid chains. Finally, CTL1-deficient Schwann cells are significantly impaired in their ability to myelinate neurites in vitro. To our knowledge, this is the first demonstration of a bona fide cell adhesion molecule, NECL4, regulating choline homeostasis and lipid biogenesis. Phosphatidylcholines are major myelin phospholipids, and several phosphorylated phosphatidylinositol species are known to regulate key aspects of peripheral myelination. Furthermore, the biophysical properties imparted to plasma membranes are regulated by fatty acid chain profiles. Therefore, it will be important to translate these in vitro observations to in vivo studies of NECL4 and CTL1-deficient mice.

Cadm3 (Necl-1) interferes with the activation of the PI3 kinase/Akt signaling cascade and inhibits Schwann cell myelination in vitro.

Axo‐glial interactions are critical for myelination and the domain organization of myelinated fibers. Cell adhesion molecules belonging to the Cadm family, and in particular Cadm3 (axonal) and its heterophilic binding partner Cadm4 (Schwann cell), mediate these interactions along the internode. Using targeted shRNA‐mediated knockdown, we show that the removal of axonal Cadm3 promotes Schwann cell myelination in the in vitro DRG neuron/Schwann cell myelinating system. Conversely, over‐expressing Cadm3 on the surface of DRG neuron axons results in an almost complete inability by Schwann cells to form myelin segments. Axons of superior cervical ganglion (SCG) neurons, which do not normally support the formation of myelin segments by Schwann cells, express higher levels of Cadm3 compared to DRG neurons. Knocking down Cadm3 in SCG neurons promotes myelination. Finally, the extracellular domain of Cadm3 interferes in a dose‐dependent manner with the activation of ErbB3 and of the pro‐myelinating PI3K/Akt pathway, but does not interfere with the activation of the Mek/Erk1/2 pathway. While not in direct contradiction, these in vitro results shed lights on the apparent lack of phenotype that was reported from in vivo studies of Cadm3−/− mice. Our results suggest that Cadm3 may act as a negative regulator of PNS myelination, potentially through the selective regulation of the signaling cascades activated in Schwann cells by axonal contact, and in particular by type III Nrg‐1. Further analyses of peripheral nerves in the Cadm−/− mice will be needed to determine the exact role of axonal Cadm3 in PNS myelination. GLIA 2016;64:2247–2262