What Is the Optimal Value of the g-Ratio for Myelinated Fibers in the Rat CNS? A Theoretical Approach

Background

The biological process underlying axonal myelination is complex and often prone to injury and disease. The ratio of the inner axonal diameter to the total outer diameter or g-ratio is widely utilized as a functional and structural index of optimal axonal myelination. Based on the speed of fiber conduction, Rushton was the first to derive a theoretical estimate of the optimal g-ratio of 0.6 [1]. This theoretical limit nicely explains the experimental data for myelinated axons obtained for some peripheral fibers but appears significantly lower than that found for CNS fibers. This is, however, hardly surprising given that in the CNS, axonal myelination must achieve multiple goals including reducing conduction delays, promoting conduction fidelity, lowering energy costs, and saving space.

Methodology/Principal Findings

In this study we explore the notion that a balanced set-point can be achieved at a functional level as the micro-structure of individual axons becomes optimized, particularly for the central system where axons tend to be smaller and their myelin sheath thinner. We used an intuitive yet novel theoretical approach based on the fundamental biophysical properties describing axonal structure and function to show that an optimal g-ratio can be defined for the central nervous system (≈0.77). Furthermore, by reducing the influence of volume constraints on structural design by about 40%, this approach can also predict the g-ratio observed in some peripheral fibers (≈0.6).

Conclusions/Significance

These results support the notion of optimization theory in nervous system design and construction and may also help explain why the central and peripheral systems have evolved different g-ratios as a result of volume constraints.     

Combinatorial lentiviral gene delivery of pro-oligodendrogenic factors for improving myelination of regenerating axons after spinal cord injury

Spinal cord injury (SCI) results in paralysis below the injury and strategies are being developed that support axonal regrowth, yet recovery lags, in part, because many axons are not remyelinated. Herein, we investigated strategies to increase myelination of regenerating axons by overexpression of platelet-derived growth factor (PDGF)-AA and noggin either alone or in combination in a mouse SCI model. Noggin and PDGF-AA have been identified as factors that enhance recruitment and differentiation of endogenous progenitors to promote myelination. Lentivirus encoding for these factors was delivered from a multichannel bridge, which we have previously shown creates a permissive environment and supports robust axonal growth through channels. The combination of noggin+PDGF enhanced total myelination of regenerating axons relative to either factor alone, and importantly, enhanced functional recovery relative to the control condition. The increase in myelination was consistent with an increase in oligodendrocyte-derived myelin, which was also associated with a greater density of cells of an oligodendroglial lineage relative to each factor individually and control conditions. These results suggest enhanced myelination of regenerating axons by noggin+PDGF that act on oligodendrocyte-lineage cells post-SCI, which ultimately led to improved functional outcomes.     

Gpr126 is essential for peripheral nerve development and myelination in mammals

In peripheral nerves, Schwann cells form the myelin sheath that insulates axons and allows rapid propagation of action potentials. Although a number of regulators of Schwann cell development are known, the signaling pathways that control myelination are incompletely understood. In this study, we show that Gpr126 is essential for myelination and other aspects of peripheral nerve development in mammals. A mutation in Gpr126 causes a severe congenital hypomyelinating peripheral neuropathy in mice, and expression of differentiated Schwann cell markers, including Pou3f1, Egr2, myelin protein zero and myelin basic protein, is reduced. Ultrastructural studies of Gpr126-/- mice showed that axonal sorting by Schwann cells is delayed, Remak bundles (non-myelinating Schwann cells associated with small caliber axons) are not observed, and Schwann cells are ultimately arrested at the promyelinating stage. Additionally, ectopic perineurial fibroblasts form aberrant fascicles throughout the endoneurium of the mutant sciatic nerve. This analysis shows that Gpr126 is required for Schwann cell myelination in mammals, and defines new roles for Gpr126 in axonal sorting, formation of mature non-myelinating Schwann cells and organization of the perineurium.     

ErbB2 directly activates the exchange factor Dock7 to promote Schwann cell migration

The cellular events that precede myelination in the peripheral nervous system require rapid and dynamic morphological changes in the Schwann cell. These events are thought to be mainly controlled by axonal signals. But how signals on the axons are coordinately organized and transduced to promote proliferation, migration, radial sorting, and myelination is unknown. We describe that the axonal signal neuregulin-1 (NRG1) controls Schwann cell migration via activation of the atypical Dock180-related guanine nucleotide exchange factor (GEF) Dock7 and subsequent activation of the Rho guanine triphosphatases (GTPases) Rac1 and Cdc42 and the downstream c-Jun N-terminal kinase. We show that the NRG1 receptor ErbB2 directly binds and activates Dock7 by phosphorylating Tyr-1118. Dock7 knockdown, or expression of Dock7 harboring the Tyr-1118-to-Phe mutation in Schwann cells, attenuates the effects of NRG1. Thus, Dock7 functions as an intracellular substrate for ErbB2 to promote Schwann cell migration. This provides an unanticipated mechanism through which ligand-dependent tyrosine phosphorylation can trigger the activation of Rho GTPase-GEFs of the Dock180 family.     

Beta1 integrin activates Rac1 in Schwann cells to generate radial lamellae during axonal sorting and myelination

Myelin is a multispiraled extension of glial membrane that surrounds axons. How glia extend a surface many-fold larger than their body is poorly understood. Schwann cells are peripheral glia and insert radial cytoplasmic extensions into bundles of axons to sort, ensheath, and myelinate them. Laminins and beta1 integrins are required for axonal sorting, but the downstream signals are largely unknown. We show that Schwann cells devoid of beta1 integrin migrate to and elongate on axons but cannot extend radial lamellae of cytoplasm, similar to cells with low Rac1 activation. Accordingly, active Rac1 is decreased in beta1 integrin-null nerves, inhibiting Rac1 activity decreases radial lamellae in Schwann cells, and ablating Rac1 in Schwann cells of transgenic mice delays axonal sorting and impairs myelination. Finally, expressing active Rac1 in beta1 integrin-null nerves improves sorting. Thus, increased activation of Rac1 by beta1 integrins allows Schwann cells to switch from migration/elongation to the extension of radial membranes required for axonal sorting and myelination.     

Axonal regulation of myelination by neuregulin 1

Neuregulins comprise a family of epidermal growth factor-like ligands that interact with ErbB receptor tyrosine kinases to control many aspects of neural development. One of the most dramatic effects of neuregulin-1 is on glial cell differentiation. The membrane-bound neuregulin-1 type III isoform is an axonal ligand for glial ErbB receptors that regulates the early Schwann cell lineage, including the generation of precursors. Recent studies have shown that the amount of neuregulin-1 type III expressed on axons also dictates the glial phenotype, with a threshold level triggering Schwann cell myelination. Remarkably, neuregulin-1 type III also regulates Schwann cell membrane growth to adjust myelin sheath thickness to match axon caliber precisely. Whether this signaling system operates in central nervous system myelination remains an open question of major importance for human demyelinating diseases.     

Axonal cell-adhesion molecule L1 in CNS myelination

Of the axonal signals influencing myelination, adhesion molecules expressed at the axonal surface are strong candidates to mediate interactions between myelinating cells and axons. The recognition cell-adhesion molecule L1, a member of the immunoglobulin superfamily has been shown to play important roles in neuronal migration and survival, and in PNS myelination. We have investigated the role of axonally expressed L1 in CNS myelination. In co-cultures of myelinating oligodendrocytes and neurons derived from murine brain, we demonstrate that, before myelination, L1 immunoreactivity is confined to neurites. After myelination commences, L1 expression is downregulated on myelinated axons and adjacent, but not yet myelinated, internodes.Interfering with L1 before the onset of myelination, by adding either anti-L1 antibody or L1-Fc fusion proteins to the culture medium, inhibits myelination. In addition, in purified cultures of oligodendrocytes, L1-Fc fusion protein prevents lysophosphatidic acid-induced activation of the mitogen-activated kinase (MAP)-kinase pathway. Together, our data indicate that L1 is involved in the initiation of CNS myelination, and that this effect might involve the dephosphorylation of oligodendroglial phosphoproteins.     

Local ERM activation and dynamic growth cones at Schwann cell tips implicated in efficient formation of nodes of Ranvier

Nodes of Ranvier are specialized, highly polarized axonal domains crucial to the propagation of saltatory action potentials. In the peripheral nervous system, axo-glial cell contacts have been implicated in Schwann cell (SC) differentiation and formation of the nodes of Ranvier. SC microvilli establish axonal contact at mature nodes, and their components have been observed to localize early to sites of developing nodes. However, a role for these contacts in node formation remains controversial.Using a myelinating explant culture system, we have observed that SCs reorganize and polarize microvillar components, such as the ezrin-binding phosphoprotein 50 kD/regulatory cofactor of the sodium-hydrogen exchanger isoform 3 (NHERF-1), actin, and the activated ezrin, radixin, and moesin family proteins before myelination in response to inductive signals. These components are targeted to the SC distal tips where live cell imaging reveals novel, dynamic growth cone-like behavior. Furthermore, localized activation of the Rho signaling pathway at SC tips gives rise to these microvillar component-enriched "caps" and influences the efficiency of node formation.     

A culture system to study oligodendrocyte myelination processes using engineered nanofibers

Current methods for studying central nervous system myelination necessitate permissive axonal substrates conducive to myelin wrapping by oligodendrocytes. We have developed a neuron-free culture system in which electron-spun nanofibers of varying sizes substitute for axons as a substrate for oligodendrocyte myelination, thereby allowing manipulation of the biophysical elements of axonal-oligodendroglial interactions. To investigate axonal regulation of myelination, this system effectively uncouples the role of molecular (inductive) cues from that of biophysical properties of the axon. We use this method to uncover the causation and sufficiency of fiber diameter in the initiation of concentric wrapping by rat oligodendrocytes. We also show that oligodendrocyte precursor cells display sensitivity to the biophysical properties of fiber diameter and initiate membrane ensheathment before differentiation. The use of nanofiber scaffolds will enable screening for potential therapeutic agents that promote oligodendrocyte differentiation and myelination and will also provide valuable insight into the processes involved in remyelination.     

How Histone Deacetylases Control Myelination

Myelinated axons are a beautiful example of symbiotic interactions between two cell types: Myelinating glial cells organize axonal membranes and build their myelin sheaths to allow fast action potential conduction, while axons regulate myelination and enhance the survival of myelinating cells. Axonal demyelination, occurring in neurodegenerative diseases or after a nerve injury, results in severe motor and/or mental disabilities. Thus, understanding how the myelination process is induced, regulated, and maintained is crucial to develop new therapeutic strategies for regeneration in the nervous system. Epigenetic regulation has recently been recognized as a fundamental contributing player. In this review, we focus on the central mechanisms of gene regulation mediated by histone deacetylation and other key functions of histone deacetylases in Schwann cells and oligodendrocytes, the myelinating glia of the peripheral and central nervous systems.     

cAMP and Schwann cells promote axonal growth and functional recovery after spinal cord injury

Central neurons regenerate axons if a permissive environment is provided; after spinal cord injury, however, inhibitory molecules are present that make the local environment nonpermissive. A promising new strategy for inducing neurons to overcome inhibitory signals is to activate cAMP signaling. Here we show that cAMP levels fall in the rostral spinal cord, sensorimotor cortex and brainstem after spinal cord contusion. Inhibition of cAMP hydrolysis by the phosphodiesterase IV inhibitor rolipram prevents this decrease and when combined with Schwann cell grafts promotes significant supraspinal and proprioceptive axon sparing and myelination. Furthermore, combining rolipram with an injection of db-cAMP near the graft not only prevents the drop in cAMP levels but increases them above those in uninjured controls. This further enhances axonal sparing and myelination, promotes growth of serotonergic fibers into and beyond grafts, and significantly improves locomotion. These findings show that cAMP levels are key for protection, growth and myelination of injured CNS axons in vivo and recovery of function.     

ErbB receptors and the development of the nervous system

Tyrosine kinase receptors and their ligands allow communication between cells in the developing and adult organism. An extensive line of research has revealed that ‘neuregulins’, a family of EGF-like factors that signal via ErbB receptors, are used frequently for cell communication during nervous system development, and control a spectacular spectrum of developmental processes. For instance, during development of the peripheral nervous system, Schwann cells require neuronally-produced neuregulin (Nrg1) for growth, migration and myelination, neural crest cells rely on mesenchymally-generated Nrg1 signals for migration, while muscle requires neuronally-produced Nrg1 for the differentiation of a muscle spindle. In the central nervous system, neuregulin signals allow cells to act as guideposts or as barriers for axons during pathfinding. Neuregulin signals are also important in other organs, but the nervous system functions have received recently considerable attention due to the finding that particular haplotypes of Nrg1 and ErbB4 predispose to schizophrenia. Understanding the neuregulin signaling system can thus contribute to define causes of this devastating mental disorder.     

Insights on the role of adhesion related molecules on stem cells

The development of the peripheral nervous system (PNS) is a highly dynamic process, during which motor and sensory axons innervate distal targets, such as skeletal muscles and skin. Axonal function depends critically on support from Schwann cells, the main glial cell type in the PNS. Schwann cells originate from the neural crest, migrate along outgrowing axons and associate with axons along their entire length prior to ensheathment or myelination. How axonal growth and the migration of Schwann cells is coordinated at the level of reciprocal axon-glial signaling is the fascinating subject of ongoing research. Neuregulin-1 (NRG1) type III, an axonal membrane-bound ligand for receptor tyrosine kinases of the ErbB family, acts as a “master regulator” of peripheral myelination. In addition, NRG1-ErbB signaling directs the development of the Schwann cell lineage and regulates the proliferation and survival of Schwann cells. Studies in zebrafish have identified a direct role of NRG1 type III in Schwann cell migration, but to what extend NRG1 serves a similar function in the mammalian PNS is not clear. We have employed a mouse superior cervical ganglion explant culture system, in which the migration of endogenous Schwann cells along outgrowing axons can be visualized by time-lapse imaging. Using this approach, we found that NRG1 type III-ErbB signaling regulates the colonization of distal axonal segments by Schwann cells. However, our data suggest an indirect effect of NRG1 type III-ErbB signaling via the support of Schwann cell survival in proximal axonal regions rather than a direct effect on Schwann cell motility.     

LINGO-1, a transmembrane signaling protein, inhibits oligodendrocyte differentiation and myelination through intercellular self-interactions

Overcoming remyelination failure is a major goal of new therapies for demyelinating diseases like multiple sclerosis. LINGO-1, a key negative regulator of myelination, is a transmembrane signaling protein expressed in both neurons and oligodendrocytes. In neurons, LINGO-1 is an integral component of the Nogo receptor complex, which inhibits axonal growth via RhoA. Because the only ligand-binding subunit of this complex, the Nogo receptor, is absent in oligodendrocytes, the extracellular signals that inhibit myelination through a LINGO-1-mediated mechanism are unknown. Here we show that LINGO-1 inhibits oligodendrocyte terminal differentiation through intercellular interactions and is capable of a self-association in trans. Consistent with previous reports, overexpression of full-length LINGO-1 inhibited differentiation of oligodendrocyte precursor cells (OPCs). Unexpectedly, treatment with a soluble recombinant LINGO-1 ectodomain also had an inhibitory effect on OPCs and decreased myelinated axonal segments in cocultures with neurons from dorsal root ganglia. We demonstrated LINGO-1-mediated inhibition of OPCs through intercellular signaling by using a surface-bound LINGO-1 construct expressed ectopically in astrocytes. Further investigation showed that the soluble LINGO-1 ectodomain can interact with itself in trans by binding to CHO cells expressing full-length LINGO-1. Finally, we observed that soluble LINGO-1 could activate RhoA in OPCs. We propose that LINGO-1 acts as both a ligand and a receptor and that the mechanism by which it negatively regulates OPC differentiation and myelination is mediated by a homophilic intercellular interaction. Disruption of this protein-protein interaction could lead to a decrease of LINGO-1 inhibition and an increase in myelination.     

Integrin-linked kinase is required for laminin-2-induced oligodendrocyte cell spreading and CNS myelination

Early steps in myelination in the central nervous system (CNS) include a specialized and extreme form of cell spreading in which oligodendrocytes extend large lamellae that spiral around axons to form myelin. Recent studies have demonstrated that laminin-2 (LN-2; alpha2beta1gamma1) stimulates oligodendrocytes to extend elaborate membrane sheets in vitro (cell spreading), mediated by integrin alpha6beta1. Although a congenital LN-2 deficiency in humans is associated with CNS white matter changes, LN-2-deficient (dy/dy) mice have shown abnormalities primarily within the peripheral nervous system. Here, we demonstrate a critical role for LN-2 in CNS myelination by showing that dy/dy mice have quantitative and morphologic defects in CNS myelin. We have defined the molecular pathway through which LN-2 signals oligodendrocyte cell spreading by demonstrating requirements for phosphoinositide 3-kinase activity and integrin-linked kinase (ILK). Interaction of oligodendrocytes with LN-2 stimulates ILK activity. A dominant negative ILK inhibits LN-2-induced myelinlike membrane formation. A critical component of the myelination signaling cascade includes LN-2 and integrin signals through ILK.     

Relationship between myelin production and dopamine synthesis in the PKU mouse brain

Phenylketonuria is caused by specific mutations in the phenylalanine hydroxylase gene and is characterized by elevated blood phenylalanine levels, hypomyelination in forebrain structures, reduced dopamine levels, and cognitive difficulties. To determine whether brain tyrosine levels and/or myelination play a role in the up-regulation of dopamine, phenylketonuric mice were placed on a low phenylalanine diet for 4 weeks and as blood phenylalanine levels dropped to normal, the relationships between phenylalanine, tyrosine, dopamine, myelin proteins, and axonal proteins in frontal cortex and striatum were determined using gas chromatography mass spectrometry, histology, and western blotting techniques. Blood phenylalanine rapidly decreased from an eight-fold elevation to near control levels, and blood tyrosine gradually rose from about 50% to near normal values. In frontal cortex and striatum, phenylalanine levels dropped to 2- and 1.5-fold elevations above control, respectively, and tyrosine levels increased but remained less than 70% of control in both structures. In frontal cortex, increases in dopamine and myelin basic protein occurred in a similar biphasic pattern, reaching near normal levels by week 4. In striatum, dopamine and MBP dramatically increased to near normal levels in the first week. Myelination was confirmed histologically and by western blot quantification of phosphorylated neurofilaments. In summary, our results showed: (i) an increase in dopamine despite low brain tyrosine levels and (ii) similar recovery patterns for myelination and dopamine. Since myelin/axonal interactions trigger signaling pathways that result in axonal maturation, we speculate that this interaction also may trigger signals that up-regulate neurotransmitter synthesis.