Requirement of cAMP signaling for Schwann cell differentiation restricts the onset of myelination.
ABSTRACT: Isolated Schwann cells (SCs) respond to cAMP elevation by adopting a differentiated post-mitotic state that exhibits high levels of Krox-20, a transcriptional enhancer of myelination, and mature SC markers such as the myelin lipid galactocerebroside (O1). To address how cAMP controls myelination, we performed a series of cell culture experiments which compared the differentiating responses of isolated and axon-related SCs to cAMP analogs and ascorbate, a known inducer of axon ensheathment, basal lamina formation and myelination. In axon-related SCs, cAMP induced the expression of Krox-20 and O1 without a concomitant increase in the expression of myelin basic protein (MBP) and without promoting axon ensheathment, collagen synthesis or basal lamina assembly. When cAMP was provided together with ascorbate, a dramatic enhancement of MBP expression occurred, indicating that cAMP primes SCs to form myelin only under conditions supportive of basal lamina formation. Experiments using a combination of cell permeable cAMP analogs and type-selective adenylyl cyclase (AC) agonists and antagonists revealed that selective transmembrane AC (tmAC) activation with forskolin was not sufficient for full SC differentiation and that the attainment of an O1 positive state also relied on the activity of the soluble AC (sAC), a bicarbonate sensor that is insensitive to forskolin and GPCR activation. Pharmacological and immunological evidence indicated that SCs expressed sAC and that sAC activity was required for morphological differentiation and the expression of myelin markers such as O1 and protein zero. To conclude, our data indicates that cAMP did not directly drive myelination but rather the transition into an O1 positive state, which is perhaps the most critical cAMP-dependent rate limiting step for the onset of myelination. The temporally restricted role of cAMP in inducing differentiation independently of basal lamina formation provides a clear example of the uncoupling of signals controlling differentiation and myelination in SCs.
Project description:Signaling through cyclic AMP (cAMP) has been implicated in the regulation of Schwann cell (SC) proliferation and differentiation. In quiescent SCs, elevation of cAMP promotes the expression of proteins associated with myelination such as Krox-20 and P0, and downregulation of markers associated with the non-myelinating SC phenotype. We have previously shown that the motor protein myosin II is required for the establishment of normal SC-axon interactions, differentiation and myelination, however, the mechanisms behind these effects are unknown. Here we report that the levels and activity of myosin light chain kinase (MLCK), an enzyme that regulates MLC phosphorylation in non-muscle cells, are dramatically downregulated in SCs after cAMP treatment, in a similar pattern to that of c-Jun, a known inhibitor of myelination. Knockdown of MLCK in SCs mimics the effect of cAMP elevation, inducing plasma membrane expansion and expression of Krox-20 and myelin proteins. Despite activation of myelin gene transcription these cells fail to make compact myelin when placed in contact with axons. Our data indicate that myosin II activity is differentially regulated at various stages during myelination and that in the absence of MLCK the processes of SC differentiation and compact myelin assembly are uncoupled.
Project description:This study was undertaken to examine the bioactivity, specificity, and reversibility of lithium's action on the growth, survival, proliferation, and differentiation of cultured Schwann cells (SCs). In isolated SCs, lithium promoted a state of cell cycle arrest that featured extensive cell enlargement and c-Jun downregulation in the absence of increased expression of myelin-associated markers. In addition, lithium effectively prevented mitogen-induced S-phase entry without impairing cell viability. When lithium was administered together with differentiating concentrations of cyclic adenosine monophosphate (cAMP) analogs, a dramatic inhibition of the expression of the master regulator of myelination Krox-20 was observed. Likewise, lithium antagonized the cAMP-dependent expression of various myelin markers such as protein zero, periaxin, and galactocerebroside and allowed SCs to maintain high levels of expression of immature SC markers even in the presence of high levels of cAMP and low levels of c-Jun. Most importantly, the inhibitory action of lithium on SC proliferation and differentiation was shown to be dose dependent, specific, and reversible upon removal of lithium compounds. In SC-neuron cultures, lithium suppressed myelin sheath formation while preserving axonal integrity, SC-axon contact, and basal lamina formation. Lithium was unique in its ability to prevent the onset of myelination without promoting myelin degradation or SC dedifferentiation. To conclude, our results underscored an unexpected antagonistic action of lithium on SC mitogenesis and myelin gene expression. We suggest that lithium represents an attractive pharmacological agent to safely and reversibly suppress the onset of SC proliferation, differentiation, and myelination while maintaining the integrity of pre-existing myelinated fibers.
Project description:Mature Schwann cells (SCs) retain dedifferentiation potential throughout adulthood. Still, how dedifferentiation occurs remains uncertain. Results from a variety of cell-based assays using in vitro cultured cAMP-differentiated and myelinating SCs revealed the existence of a novel dedifferentiating activity expressed on the surface of dorsal root ganglion (DRG) axons. This activity had the capacity to prevent SC differentiation and elicit dedifferentiation through direct SC-axon contact. Evidence is provided showing that a rapid loss of myelinating SC markers concomitant to proliferation occurred even in the presence of elevated cAMP, a signal that is required to drive and maintain a differentiated state. The dedifferentiating activity was a membrane-bound protein found exclusively in DRG neurons, as judged by its subcellular partitioning, sensitivity to proteolytic degradation and cell-type specificity, and remained active even after disruption of cellular organization. It differed from the membrane-anchored neuregulin-1 isoforms that are responsible for axon contact-induced SC proliferation and exerted its action independently of mitogenic signaling emanating from receptor tyrosine kinases and mitogen-activated protein kinases such as ERK and JNK. Interestingly, dedifferentiation occurred without concomitant changes in the expression of Krox-20, a transcriptional enhancer of myelination, and c-Jun, an inhibitor of myelination. In sum, our data indicated the existence of cell surface axon-derived signals that override pro-differentiating cues, drive dedifferentiation and allow SCs to proliferate in response to axonal mitogens. This axonal signal may negatively regulate myelination at the onset or reversal of the differentiated state. GLIA 2017;65:851-863.
Project description:Myelin plays a crucial role in axon function recovery following nerve damage, and the interaction between Schwann cells (SCs) and regenerating axons profoundly affects myelin formation. Eph receptor A4 (EphA4), a member of the Eph tyrosine kinase receptor family, regulates cell-cell interactions via its ligand ephrins. However, our current knowledge on how EphA4 regulates the formation of myelin sheaths remains limited. In order to explore the roles of EphA4 in myelination in the peripheral nervous system, we used a combination of (1) a co-culture model of dorsal root ganglion (DRG) explants and SCs, (2) a SC differentiation model induced by db-cAMP, and (3) a regeneration model of crushed sciatic nerves in rats. Our results demonstrated that EphA4 inhibited myelination by inhibiting SC differentiation and facilitating SC proliferation in vitro. The in vivo experiments revealed that EphA4 expression in SCs is upregulated following nerve crush injury and then downregulated during remyelination. Moreover, silencing of EphA4 by siRNA or overexpression of EphA4 by genetic manipulation can accelerate or slow down nerve remyelination in crushed sciatic nerves. Taken together, our results suggest that EphA4 may negatively regulate myelination by abrogating SC differentiation.
Project description:This study comprehensively addresses the phenotype, function, and whole transcriptome of primary human and rodent Schwann cells (SCs) and highlights key species-specific features beyond the expected donor variability that account for the differential ability of human SCs to proliferate, differentiate, and interact with axons in vitro. Contrary to rat SCs, human SCs were insensitive to mitogenic factors other than neuregulin and presented phenotypic variants at various stages of differentiation, along with a mixture of proliferating and senescent cells, under optimal growth-promoting conditions. The responses of human SCs to cAMP-induced differentiation featured morphological changes and cell cycle exit without a concomitant increase in myelin-related proteins and lipids. Human SCs efficiently extended processes along those of other SCs (human or rat) but failed to do so when placed in co-culture with sensory neurons under conditions supportive of myelination. Indeed, axon contact-dependent human SC alignment, proliferation, and differentiation were not observed and could not be overcome by growth factor supplementation. Strikingly, RNA-seq data revealed that ~?44 of the transcriptome contained differentially expressed genes in human and rat SCs. A bioinformatics approach further highlighted that representative SC-specific transcripts encoding myelin-related and axon growth-promoting proteins were significantly affected and that a deficient expression of key transducers of cAMP and adhesion signaling explained the fairly limited potential of human SCs to differentiate and respond to axonal cues. These results confirmed the significance of combining traditional bioassays and high-resolution genomics methods to characterize human SCs and identify genes predictive of cell function and therapeutic value.
Project description:Genetically modified mice have been a major source of information about the molecular control of Schwann-cell myelin formation, and the role of ?-neuregulin 1 (NRG1) in this process in vivo. In vitro, on the other hand, Schwann cells from rats have been used in most analyses of the signaling pathways involved in myelination. To correlate more effectively in vivo and in vitro data, we used purified cultures of mouse Schwann cells in addition to rat Schwann cells to examine two important myelin-related signals, cyclic adenosine monophosphate (cAMP), and NRG1 and to determine whether they interact to control myelin differentiation. We find that in mouse Schwann cells, neither cAMP nor NRG1, when used separately, induced markers of myelin differentiation. When combined, however, they induced strong protein expression of the myelin markers, Krox-20 and P(0) . Importantly, the level of cAMP signaling was crucial in switching NRG1 from a proliferative signal to a myelin differentiation signal. Also in cultured rat Schwann cells, NRG1 promoted cAMP-induced Krox-20 and P(0) expression. Finally, we found that cAMP/NRG1-induced Schwann-cell differentiation required the activity of the cAMP response element binding family of transcription factors in both mouse and rat cells. These observations reconcile observations in vivo and on neuron-Schwann-cell cultures with studies on purified Schwann cells. They demonstrate unambiguously the promyelin effects of NRG1 in purified cells, and they show that the cAMP pathway determines whether NRG1 drives proliferation or induces myelin differentiation.
Project description:Vesicular release from neurons promotes myelin sheath growth on axons. Oligodendrocytes express proteins that allow dendrites to respond to vesicular release at synapses, suggesting that axon-myelin contacts use similar communication mechanisms as synapses to form myelin sheaths. To test this, we used fusion proteins to track synaptic vesicle localization and membrane fusion in zebrafish during developmental myelination and investigated expression and localization of PSD95, a dendritic post-synaptic protein, within oligodendrocytes. Synaptic vesicles accumulate and exocytose at ensheathment sites with variable patterning and most sheaths localize PSD95 with patterning similar to exocytosis site location. Disruption of candidate PDZ-binding transsynaptic adhesion proteins in oligodendrocytes cause variable effects on sheath length and number. One candidate, Cadm1b, localizes to myelin sheaths where both PDZ binding and extracellular adhesion to axons mediate sheath growth. Our work raises the possibility that axon-glial communication contributes to myelin plasticity, providing new targets for mechanistic unraveling of developmental myelination.
Project description:In the nervous system, myelination of axons enables rapid impulse conduction and is a specialized function of glial cells. Myelinating glia are the last cell type to emerge in the evolution of vertebrate nervous systems, presumably in ancient jawed vertebrates (gnathostomata) because jawless vertebrates (agnathans) lack myelin. We have hypothesized that, in these unmyelinated species, evolutionary progenitors of myelinating cells must have existed that should still be present in contemporary agnathan species. Here, we used advanced electron microscopic techniques to reveal axon-glia interactions in the sea lamprey Petromyzon marinus By quantitative assessment of the spinal cord and the peripheral lateral line nerve, we observed a marked maturation-dependent growth of axonal calibers. In peripheral nerves, all axons are ensheathed by glial cells either in bundles or, when larger than the threshold caliber of 3 ?m, individually. The ensheathing glia are covered by a basal lamina and express SoxE-transcription factors, features of mammalian Remak-type Schwann cells. In larval lamprey, the ensheathment of peripheral axons leaves gaps that are closed in adults. CNS axons are also covered to a considerable extent by glial processes, which contain a high density of intermediate filaments, glycogen particles, large lipid droplets, and desmosomes, similar to mammalian astrocytes. Indeed, by in situ hybridization, these glial cells express the astrocyte marker Aldh1l1 Specimens were of unknown sex. Our observations imply that radial sorting, ensheathment, and presumably also metabolic support of axons are ancient functions of glial cells that predate the evolutionary emergence of myelin in jawed vertebrates.SIGNIFICANCE STATEMENT We used current electron microscopy techniques to examine axon-glia units in a nonmyelinated vertebrate species, the sea lamprey. In the PNS, lamprey axons are fully ensheathed either individually or in bundles by cells ortholog to Schwann cells. In the CNS, axons associate with astrocyte orthologs, which contain glycogen and lipid droplets. We suggest that ensheathment, radial sorting, and metabolic support of axons by glial cells predate the evolutionary emergence of myelin in ancient jawed vertebrates.
Project description:Oligodendrocyte-axon contact is mediated by several cell adhesion molecules (CAMs) that are positioned at distinct sites along the myelin unit, yet their role during myelination remains unclear. Cadm4 and its axonal receptors, Cadm2 and Cadm3, as well as myelin-associated glycoprotein (MAG), are enriched at the internodes below the compact myelin, whereas NF155, which binds the axonal Caspr/contactin complex, is located at the paranodal junction that is formed between the axon and the terminal loops of the myelin sheath. Here we report that Cadm4-, MAG-, and Caspr-mediated adhesion cooperate during myelin membrane ensheathment. Genetic deletion of either Cadm4 and MAG or Cadm4 and Caspr resulted in the formation of multimyelinated axons due to overgrowth of the myelin away from the axon and the forming paranodal junction. Consequently, these mice displayed paranodal loops either above or underneath compact myelin. Our results demonstrate that accurate placement of the myelin sheath by oligodendrocytes requires the coordinated action of internodal and paranodal CAMs.
Project description:Myelin ensheathes axons to allow rapid propagation of action potentials and proper nervous system function. In the peripheral nervous system, Schwann cells (SCs) radially sort axons into a 1:1 relationship before wrapping an axonal segment to form myelin. SC myelination requires the adhesion G protein-coupled receptor GPR126, which undergoes autoproteolytic cleavage into an N-terminal fragment (NTF) and a seven-transmembrane-containing C-terminal fragment (CTF). Here we show that GPR126 has domain-specific functions in SC development whereby the NTF is necessary and sufficient for axon sorting, whereas the CTF promotes wrapping through cAMP elevation. These biphasic roles of GPR126 are governed by interactions with Laminin-211, which we define as a novel ligand for GPR126 that modulates receptor signaling via a tethered agonist. Our work suggests a model in which Laminin-211 mediates GPR126-induced cAMP levels to control early and late stages of SC development.