Akt Regulates Axon Wrapping and Myelin Sheath Thickness in the PNS.

The signaling pathways that regulate myelination in the PNS remain poorly understood. Phosphatidylinositol-4,5-bisphosphate 3-kinase 1A, activated in Schwann cells by neuregulin and the extracellular matrix, has an essential role in the early events of myelination. Akt/PKB, a key effector of phosphatidylinositol-4,5-bisphosphate 3-kinase 1A, was previously implicated in CNS, but not PNS myelination. Here we demonstrate that Akt plays a crucial role in axon ensheathment and in the regulation of myelin sheath thickness in the PNS. Pharmacological inhibition of Akt in DRG neuron-Schwann cell cocultures dramatically decreased MBP and P0 levels and myelin sheath formation without affecting expression of Krox20/Egr2, a key transcriptional regulator of myelination. Conversely, expression of an activated form of Akt in purified Schwann cells increased expression of myelin proteins, but not Krox20/Egr2, and the levels of activated Rac1. Transgenic mice expressing a membrane-targeted, activated form of Akt under control of the 2',3'-cyclic nucleotide 3'-phosphodiesterase promoter, exhibited thicker PNS and CNS myelin sheaths, and PNS myelin abnormalities, such as tomacula and myelin infoldings/outfoldings, centered around the paranodes and Schmidt Lanterman incisures. These effects were corrected by rapamycin treatmentin vivo Importantly, Akt activity in the transgenic mice did not induce myelination of nonmyelinating Schwann cells in the sympathetic trunk or Remak fibers of the dorsal roots, although, in those structures, they wrapped membranes redundantly around axons. Together, our data indicate that Akt is crucial for PNS myelination driving axonal wrapping by unmyelinated and myelinated Schwann cells and enhancing myelin protein synthesis in myelinating Schwann cells. SIGNIFICANCE STATEMENT: Although the role of the key serine/threonine kinase Akt in promoting CNS myelination has been demonstrated, its role in the PNS has not been established and remains uncertain. This work reveals that Akt controls several key steps of the PNS myelination. First, its activity promotes membrane production and axonal wrapping independent of a transcriptional effect. In myelinated axons, it also enhances myelin thickness through the mTOR pathway. Finally, sustained Akt activation in Schwann cells leads to hypermyelination/dysmyelination, mimicking some features present in neuropathies, such as hereditary neuropathy with liability to pressure palsies or demyelinating forms of Charcot-Marie-Tooth disease. Together, these data demonstrate the role of Akt in regulatory mechanisms underlying axonal wrapping and myelination in the PNS.

Functionally distinct PI 3-kinase pathways regulate myelination in the peripheral nervous system.

The PI 3-kinase (PI 3-K) signaling pathway is essential for Schwann cell myelination. Here we have characterized PI 3-K effectors activated during myelination by probing myelinating cultures and developing nerves with an antibody that recognizes phosphorylated substrates for this pathway. We identified a discrete number of phospho-proteins including the S6 ribosomal protein (S6rp), which is down-regulated at the onset of myelination, and N-myc downstream-regulated gene-1 (NDRG1), which is up-regulated strikingly with myelination. We show that type III Neuregulin1 on the axon is the primary activator of S6rp, an effector of mTORC1. In contrast, laminin-2 in the extracellular matrix (ECM), signaling through the α6ß4 integrin and Sgk1 (serum and glucocorticoid-induced kinase 1), drives phosphorylation of NDRG1 in the Cajal bands of the abaxonal compartment. Unexpectedly, mice deficient in α6ß4 integrin signaling or Sgk1 exhibit hypermyelination during development. These results identify functionally and spatially distinct PI 3-K pathways: an early, pro-myelinating pathway driven by axonal Neuregulin1 and a later-acting, laminin-integrin-dependent pathway that negatively regulates myelination.

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.

Myosin II is a negative regulator of oligodendrocyte morphological differentiation.

During their development as myelinating cells, oligodendrocyte progenitors (OPC) undergo dramatic changes in the organization of their cytoskeleton. These changes involve an increase in cell branching and in lamella extension, which is important for the ability of oligodendrocytes to myelinate multiple axons in the CNS. We have previously shown that the levels of the actin-associated motor protein nonmuscle myosin II (NMII) decrease as oligodendrocyte differentiate and that inhibition of NMII activity increases branching and myelination, suggesting that NMII is a negative regulator of oligodendrocyte differentiation. In agreement with this interpretation, we have found that overexpression of NMII prevents oligodendrocyte branching and differentiation and that OPC maturation is accelerated in NMII knockout mice as shown by a significant increase in the percentage of mature MBP(+) cells. Although several pathways have been implicated in oligodendrocyte morphogenesis, their specific contribution to the regulation of NMII activity has not been directly examined. We tested the hypothesis that the activity of NMII in OPC is controlled by Fyn kinase via downregulation of RhoA-ROCK-NMII phosphorylation. We found that treatment with PP2 or knockdown of Fyn using siRNA prevents the decrease in myosin phosphorylation normally observed during OPC differentiation and that the inhibition of branching induced by overexpression of constitutively active RhoA can be reversed by treatment with Y27632 or blebbistatin. Taken together, our results demonstrate that Fyn kinase downregulates NMII activity, thus promoting oligodendrocyte morphological differentiation.

MLCK regulates Schwann cell cytoskeletal organization, differentiation and myelination.

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.

Electron tomography of paranodal septate-like junctions and the associated axonal and glial cytoskeletons in the central nervous system.

The polarized domains of myelinated axons are specifically organized to maximize the efficiency of saltatory conduction. The paranodal region is directly adjacent to the node of Ranvier and contains specialized septate-like junctions that provide adhesion between axons and glial cells and that constitute a lateral diffusion barrier for nodal components. To complement and extend earlier studies on the peripheral nervous system, electron tomography was used to image paranodal regions from the central nervous system (CNS). Our three-dimensional reconstructions revealed short filamentous linkers running directly from the septate-like junctions to neurofilaments, microfilaments, and organelles within the axon. The intercellular spacing between axons and glia was measured to be 7.4 ± 0.6 nm, over twice the value previously reported in the literature (2.5-3.0 nm). Averaging of individual junctions revealed a bifurcated structure in the intercellular space that is consistent with a dimeric complex of cell adhesion molecules composing the septate-like junction. Taken together, these findings provide new insight into the structural organization of CNS paranodes and suggest that, in addition to providing axo-glial adhesion, cytoskeletal linkage to the septate-like junctions may be required to maintain axonal domains and to regulate organelle transport in myelinated axons.

Myosin II has distinct functions in PNS and CNS myelin sheath formation.

The myelin sheath forms by the spiral wrapping of a glial membrane around the axon. The mechanisms responsible for this process are unknown but are likely to involve coordinated changes in the glial cell cytoskeleton. We have found that inhibition of myosin II, a key regulator of actin cytoskeleton dynamics, has remarkably opposite effects on myelin formation by Schwann cells (SC) and oligodendrocytes (OL). Myosin II is necessary for initial interactions between SC and axons, and its inhibition or down-regulation impairs their ability to segregate axons and elongate along them, preventing the formation of a 1:1 relationship, which is critical for peripheral nervous system myelination. In contrast, OL branching, differentiation, and myelin formation are potentiated by inhibition of myosin II. Thus, by controlling the spatial and localized activation of actin polymerization, myosin II regulates SC polarization and OL branching, and by extension their ability to form myelin. Our data indicate that the mechanisms regulating myelination in the peripheral and central nervous systems are distinct.

Type III neuregulin-1 promotes oligodendrocyte myelination.

The axonal signals that regulate oligodendrocyte myelination during development of the central nervous system (CNS) have not been established. In this study, we have examined the regulation of oligodendrocyte myelination by the type III isoform of neuregulin-1 (NRG1), a neuronal signal essential for Schwann cell differentiation and myelination. In contrast to Schwann cells, primary oligodendrocytes differentiate normally when cocultured with dorsal root ganglia (DRG) neurons deficient in type III NRG1. However, they myelinate type III NRG1-deficient neurites poorly in comparison to wild type cultures. Type III NRG1 is not sufficient to drive oligodendrocyte myelination as sympathetic neurons are not myelinated even with lentiviral-mediated expression of NRG1. Mice haploinsufficient for type III NRG1 are hypomyelinated in the brain, as evidenced by reduced amounts of myelin proteins and lipids and thinner myelin sheaths. In contrast, the optic nerve and spinal cord of heterozygotes are myelinated normally. Together, these results implicate type III NRG1 as a significant determinant of the extent of myelination in the brain and demonstrate important regional differences in the control of CNS myelination. They also indicate that oligodendrocyte myelination, but not differentiation, is promoted by axonal NRG1, underscoring important differences in the control of myelination in the CNS and peripheral nervous system (PNS).

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.

Interleukin-11 potentiates oligodendrocyte survival and maturation, and myelin formation.

Mechanisms that regulate oligodendrocyte survival and myelin formation are an intense focus of research into myelin repair in the lesions of multiple sclerosis (MS). Although demyelination and oligodendrocyte loss are pathological hallmarks of the disease, increased oligodendrocyte numbers and remyelination are frequently observed in early lesions, but these diminish as the disease course progresses. In the current study, we used a microarray-based approach to investigate genes regulating repair in MS lesions, and identified interleukin-11 (IL-11) as an astrocyte-derived factor that potentiates oligodendrocyte survival and maturation, and myelin formation. IL-11 was induced in human astrocyte cultures by the cytokines IL-1beta and TGFbeta1, which are both prominently expressed in MS plaques. In MS tissue samples, IL-11 was expressed by reactive astrocytes, with expression particularly localized at the myelinated border of both active and silent lesions. Its receptor, IL-11R alpha, was expressed by oligodendrocytes. In experiments in human cultures in vitro, IL-11R alpha localized to immature oligodendrocytes, and its expression decreased during maturation. In cultures treated with IL-11, we observed a significant increase in oligodendrocyte number, and this was associated with enhanced oligodendrocyte survival and maturation. Importantly, we also found that IL-11 treatment was associated with significantly increased myelin formation in rodent CNS cocultures. These data are the first to implicate IL-11 in oligodendrocyte viability, maturation, and myelination. We suggest that this pathway may represent a potential therapeutic target for oligodendrocyte protection and remyelination in MS.

Disrupted axo-glial junctions result in accumulation of abnormal mitochondria at nodes of ranvier.

Mitochondria and other membranous organelles are frequently enriched in the nodes and paranodes of peripheral myelinated axons, particularly those of large caliber. The physiologic role(s) of this organelle enrichment and the rheologic factors that regulate it are not well understood. Previous studies suggest that axonal transport of organelles across the nodal/paranodal region is locally regulated. In this study, we have examined the ultrastructure of myelinated axons in the sciatic nerves of mice deficient in the contactin-associated protein (Caspr), an integral junctional component. These mice, which lack the normal septate-like junctions that promote attachment of the glial (paranodal) loops to the axon, contain aberrant mitochondria in their nodal/paranodal regions. These mitochondria are typically large and swollen and occupy prominent varicosities of the nodal axolemma. In contrast, mitochondria located outside the nodal/paranodal regions of the myelinated axons appear normal. These findings suggest that paranodal junctions regulate mitochondrial transport and function in the axoplasm of the nodal/paranodal region of myelinated axons of peripheral nerves. They further implicate the paranodal junctions in playing a role, either directly or indirectly, in the local regulation of energy metabolism in the nodal region.

Neuregulin-1 type III determines the ensheathment fate of axons.

The signals that determine whether axons are ensheathed or myelinated by Schwann cells have long been elusive. We now report that threshold levels of neuregulin-1 (NRG1) type III on axons determine their ensheathment fate. Ensheathed axons express low levels whereas myelinated fibers express high levels of NRG1 type III. Sensory neurons from NRG1 type III deficient mice are poorly ensheathed and fail to myelinate; lentiviral-mediated expression of NRG1 type III rescues these defects. Expression also converts the normally unmyelinated axons of sympathetic neurons to myelination. Nerve fibers of mice haploinsufficient for NRG1 type III are disproportionately unmyelinated, aberrantly ensheathed, and hypomyelinated, with reduced conduction velocities. Type III is the sole NRG1 isoform retained at the axon surface and activates PI 3-kinase, which is required for Schwann cell myelination. These results indicate that levels of NRG1 type III, independent of axon diameter, provide a key instructive signal that determines the ensheathment fate of axons.

Integrin alpha(v)beta8-mediated activation of transforming growth factor-beta by perivascular astrocytes: an angiogenic control switch.

Brain hemorrhage is a severe complication of both neoplastic and nonneoplastic brain disease. Mice deficient in the alpha(v)beta8 integrin display defective brain vessel formation resulting in hemorrhage and perinatal death, but the mechanism of brain hemorrhage is unknown. Because the alpha(v)beta8 integrin is expressed by astrocytes and not expressed by endothelium, paracrine interactions between astrocytes and endothelial cells could contribute to the maintenance of brain vessel integrity. We have investigated the mechanisms underlying astrocytic-endothelial paracrine signaling and have found that integrin-mediated activation of transforming growth factor (TGF)-beta by astrocytes influences endothelial cell function. Thus, we identified the integrin alpha(v)beta8 in human perivascular glial cell processes surrounding developing blood vessels. Human astrocytic alpha(v)beta8 was a major cell surface receptor for latent TGF-beta, and alpha(v)beta8-dependent activation of TGF-beta was the major mechanism of TGF-beta activation in primary cultures of astrocytes or freshly dissociated fetal brain cells. This activation of TGF-beta was sufficient to inhibit endothelial migration in fibrin gels and to alter expression of genes affecting proteolytic and angiogenic pathways. Taken together, our data suggest that astrocytic alpha(v)beta8 acts as a central regulator of brain vessel homeostasis through regulation of TGF-beta activation and expression of TGF-beta-responsive genes that promote vessel differentiation and stabilization, most notably plasminogen activator inhibitor-1 and thrombospondin-1.

Rho kinase regulates schwann cell myelination and formation of associated axonal domains.

The myelin sheath forms by the spiral wrapping of a glial membrane around an axon. The mechanisms involved are poorly understood but are likely to involve coordinated changes in the glial cell cytoskeleton. Because of its key role as a regulator of the cytoskeleton, we investigated the role of Rho kinase (ROCK), a major downstream effector of Rho, in Schwann cell morphology, differentiation, and myelination. Pharmacologic inhibition of ROCK activity results in loss of microvilli and stress fibers in Schwann cell cultures and strikingly aberrant myelination in Schwann cell-neuron cocultures; there was no effect on Schwann cell proliferation or differentiation. Treated Schwann cells branch aberrantly and form multiple, small, independent myelin segments along the length of axons, each with associated nodes and paranodes. This organization partially resembles myelin formed by oligodendrocytes rather than the single long myelin sheath characteristic of Schwann cells. ROCK regulates myosin light chain phosphorylation, which is robustly, but transiently, activated at the onset of myelination. These results support a key role of Rho through its effector ROCK in coordinating the movement of the glial membrane around the axon at the onset of myelination via regulation of myosin phosphorylation and actomyosin assembly. They also indicate that the molecular machinery that promotes the wrapping of the glial membrane sheath around the axon is distributed along the entire length of the internode.

Paranodal interactions regulate expression of sodium channel subtypes and provide a diffusion barrier for the node of Ranvier.

The node of Ranvier is a distinct domain of myelinated axons that is highly enriched in sodium channels and is critical for impulse propagation. During development, the channel subtypes expressed at the node undergo a transition from Nav1.2 to Nav1.6. Specialized junctions that form between the paranodal glial membranes and axon flank the nodes and are candidates to regulate their maturation and delineate their boundaries. To investigate these roles, we characterized node development in mice deficient in contactin-associated protein (Caspr), an integral junctional component. Paranodes in these mice lack transverse bands, a hallmark of the mature junction, and exhibit progressive disruption of axon-paranodal loop interactions in the CNS. Caspr mutant mice display significant abnormalities at central nodes; components of the nodes progressively disperse along axons, and many nodes fail to mature properly, persistently expressing Nav1.2 rather than Nav1.6. In contrast, PNS nodes are only modestly longer and, although maturation is delayed, eventually all express Nav1.6. Potassium channels are aberrantly clustered in the paranodes; these clusters are lost over time in the CNS, whereas they persist in the PNS. These findings indicate that interactions of the paranodal loops with the axon promote the transition in sodium channel subtypes at CNS nodes and provide a lateral diffusion barrier that, even in the absence of transverse bands, maintains a high concentration of components at the node and the integrity of voltage-gated channel domains.