Promotion of Remyelination by Sulfasalazine in a Transgenic Zebrafish Model of Demyelination

Most of the axons in the vertebrate nervous system are surrounded by a lipid-rich membrane called myelin, which promotes rapid conduction of nerve impulses and protects the axon from being damaged. Multiple sclerosis (MS) is a chronic demyelinating disease of the CNS characterized by infiltration of immune cells and progressive damage to myelin and axons. One potential way to treat MS is to enhance the endogenous remyelination process, but at present there are no available treatments to promote remyelination in patients with demyelinating diseases.Sulfasalazine is an anti-inflammatory and immune-modulating drug that is used in rheumatology and inflammatory bowel disease. Its anti-inflammatory and immunomodulatory properties prompted us to test the ability of sulfasalazine to promote remyelination. In this study, we found that sulfasalazine promotes remyelination in the CNS of a transgenic zebrafish model of NTR/MTZ-induced demyelination. We also found that sulfasalazine treatment reduced the number of macrophages/microglia in the CNS of demyelinated zebrafish larvae, suggesting that the acceleration of remyelination is mediated by the immunomodulatory function of sulfasalazine. Our data suggest that temporal modulation of the immune response by sulfasalazine can be used to overcome MS by enhancing myelin repair and remyelination in the CNS.     

Schwann cells in the regenerating fish optic nerve: Evidence that CNS axons,not the glia,determine when myelin formation begins

Fish optic nerve fibres quickly regenerate after injury, but the onset of remyelination is delayed until they reach the brain. This recapitulates the timetable of CNS myelinogenesis during development in vertebrate animals generally, and we have used the regenerating fish optic nerve to obtain evidence that it is the axons, not the myelinating glial cells, that determine when myelin formation begins. In fish, the site of an optic nerve injury becomes remyelinated by ectopic Schwann cells of unknown origin. We allowed these cells to become established and then used them as reporters to indicate the time course of pro-myelin signalling during a further round of axonal outgrowth following a second upstream lesion. Unlike in the mammalian PNS, the ectopic Schwann cells failed to respond to axotomy and to the initial outgrowth of new optic axons. They only began to divide after the axons had reached the brain. Shortly afterwards, small numbers of Schwann cells began to leave the dividing pool and form myelin sheaths. More followed gradually, so that by 3 months remyelination was almost completed and few dividing cells were left. Moreover, remyelination occurred synchronously throughout the optic nerve, with the same time course in the pre-existing Schwann cells, the new ones that colonised the second injury, and the CNS oligodendrocytes elsewhere. The optic axons are the only common structures that could synchronise myelin formation in these disparate glial populations. The responses of the ectopic Schwann cells suggest that they are controlled by the regenerating optic axons in two consecutive steps. First, they begin to proliferate when the growing axons reach the brain. Second, they leave the cell cycle to differentiate individually at widely different times during the ensuing 2 months, during the critical period when the initial rough pattern of axon terminals in the optic tectum becomes refined into an accurate map. We suggest that each axon signals individually for myelin ensheathment once it completes this process.     

Optical measurement of conduction in single demyelinated axons

Demyelination was initiated in Xenopus sciatic nerves by an intraneural injection of lysolecithin over a 2-3-mm region. During the next week macrophages and Schwann cells removed all remaining damaged myelin by phagocytosis. Proliferating Schwann cells then began to remyelinate the axons, with the first few lamellae appearing 13 d after surgery. Action potentials were recorded optically through the use of a potential-sensitive dye. Signals could be detected both at normal nodes of Ranvier and within demyelinated segments. Before remyelination, conduction through the lesion occurred in only a small fraction of the fibers. However, in these particular cases we could demonstrate continuous (nonsaltatory) conduction at very low velocities over long (greater than one internode) lengths of demyelinated axons. We have previously found through loose patch clamp experiments that the internodal axolemma contains voltage-dependent Na+ channels at a density approximately 4% of that at the nodes. These channels alone, however, are insufficient for successful conduction past the transition point between myelinated and demyelinated regions. Small improvements in the passive cable properties of the axon, adequate for propagation at this site, can be realized through the close apposition of macrophages and Schwann cells. As the initial lamellae of myelin appear, the probability of success at the transition zone increases rapidly, though the conduction velocity through the demyelinated segment is not appreciably changed. A detailed computational model is used to test the relative roles of the internodal Na+ channels and the new extracellular layer. The results suggest a possible mechanism that may contribute to the spontaneous recovery of function often seen in demyelinating disease.     

Schwann cells responding to primary demyelination in vivo express p75NTR and c-erbB receptors: a light and electron immunohistochemical study

We have used quantitative and qualitative light microscope immunohistochemistry to examine the expression of p75NTR and c-erbB receptors in Schwann cells in a demyelinating lesion induced by the intraneural injection of lysophosphatidyl choline (LPC). We report that levels of p75NTR, c-erbB2 and c-erbB4, as assessed using image analysis of immuno-peroxidase labelled sections, and c-erbB3, as assessed by eye, increased within each lesion site soon after the initiation of myelinolysis, peaked between 5 and 8 days after induction of demyelination and fell to undetectable levels at the onset of remyelination. Pre-embedding immunoelectron microscopy confirmed that Schwann cells ensheathing demyelinated axons were p75NTR positive. Immunolabel decorated overlapping processes of neighbouring Schwann cells, suggesting that in this context p75NTR could play a role in juxtacrine signalling between reacting cells. We conclude that upregulation of p75NTR and c-erbB receptors is a constitutive Schwann cell response to an acute disruption of the axon–Schwann cell relationship.     

Fibrin inhibits peripheral nerve remyelination by regulating Schwann cell differentiation

Remyelination is a critical step for functional nerve regeneration. Here we show that fibrin deposition in the peripheral nervous system after injury is a key regulator of remyelination. After sciatic nerve crush, fibrin is deposited and its clearance correlates with remyelination. Fibrin induces phosphorylation of ERK1/2 and production of p75 NGF low-affinity receptor in Schwann cells and maintains them in a nonmyelinating state, suppresses fibronectin production, and prevents synthesis of myelin proteins. In mice depleted of fibrin(ogen), remyelination of myelinated axons is accelerated due to the faster transition of the Schwann cells to a myelinating state. Regulation of fibrin clearance and/or deposition could be a key regulatory mechanism for Schwann differentiation after nerve damage.     

Direct visualization of membrane architecture of myelinating cells in transgenic mice expressing membrane‐anchored EGFP

Myelinogenesis is a complex process that involves substantial and dynamic changes in plasma membrane architecture and myelin interaction with axons. Highly ramified processes of oligodendrocytes in the central nervous system (CNS) make axonal contact and then extrapolate to wrap around axons and form multilayer compact myelin sheathes. Currently, the mechanisms governing myelin sheath assembly and axon selection by myelinating cells are not fully understood. Here, we generated a transgenic mouse line expressing the membrane‐anchored green fluorescent protein (mEGFP) in myelinating cells, which allow live imaging of details of myelinogenesis and cellular behaviors in the nervous systems. mEGFP expression is driven by the promoter of 2'‐3'‐cyclic nucleotide 3'‐phosphodiesterase (CNP) that is expressed in the myelinating cell lineage. Robust mEGFP signals appear in the membrane processes of oligodendrocytes in the CNS and Schwann cells in the peripheral nervous system (PNS), wherein mEGFP expression defines the inner layers of myelin sheaths and Schmidt‐Lanterman incisures in adult sciatic nerves. In addition, mEGFP expression can be used to track the extent of remyelination after demyelinating injury in a toxin‐induced demyelination animal model. Taken together, the membrane‐anchored mEGFP expression in the new transgenic line would facilitate direct visualization of dynamic myelin membrane formation and assembly during development and process remodeling during remyelination after various demyelinating injuries. genesis 52:341–349, 2014. © 2014 Wiley Periodicals, Inc.     

The Na+/Ca2+ exchangers in demyelinating diseases

Intracellular [Na⁺]_i and [Ca²⁺]_i imbalance significantly contribute to neuro-axonal dysfunctions and maladaptive myelin repair or remyelination failure in chronic inflammatory demyelinating diseases such as multiple sclerosis. Progress in recent years has led to significant advances in understanding how [Ca²⁺]_i signaling network drive degeneration or remyelination of demyelinated axons.The Na⁺/Ca²⁺ exchangers (NCXs), a transmembrane protein family including three members encoded by ncx1, ncx2, and ncx3 genes, are emerging important regulators of [Na⁺]_i and [Ca²⁺]_i both in neurons and glial cells. Here we review recent advance highlighting the role of NCX exchangers in axons and myelin-forming cells, i.e. oligodendrocytes, which represent the major targets of the aberrant inflammatory attack in multiple sclerosis. The contribution of NCX subtypes to axonal pathology and myelin synthesis will be discussed. Although a definitive understanding of mechanisms regulating axonal pathology and remyelination failure in chronic demyelinating diseases is still lacking and requires further investigation, current knowledge suggest that NCX activity plays a crucial role in these processes. Defining the relative contributions of each NCX transporter in axon pathology and myelinating glia will constitute not only a major advance in understanding in detail the intricate mechanism of neurodegeneration and remyelination failure in demyelinating diseases but also will help to identify neuroprotective or remyelinating strategies targeting selective NCX exchangers as a means of treating MS.     

Remyelination of spinal cord axons by olfactory ensheathing cells and Schwann cells derived from a transgenic rat expressing alkaline phosphatase marker gene

Transplantation of cell suspensions containing olfactory ensheathing cells (OECs) has been reported to remyelinate demyelinated axons in the spinal cord with a Schwann cell (SC)-like pattern of myelination. However, questions have been raised recently as to whether OECs can form SC-like myelin. To address this issue we prepared SCs and OECs from transgenic rats in which a marker gene, human placental alkaline phosphatase (hPAP), is linked to the ubiquitously active promoter of the R26 gene. SCs were prepared from the sciatic nerve and OECs from the outer nerve-fiber layer of the olfactory bulb. Positive S100 and p75 immunostaining indicated that >95% of cells in culture displayed either SC or OEC phenotypes. Suspensions of either SCs or OECs were transplanted into an X-irradiation/ethidium bromide demyelinating lesion in the spinal cord. We observed extensive SC-like remyelination following either SC or OEC transplantation 3 weeks after injection of the cells. Alkaline phosphatase (ALP) chromagen reaction product was associated clearly with the myelin-forming cells. Thus, cell suspensions that are enriched in either SCs or OECs result in peripheral-like myelin when transplanted in vivo.     

Farnesol Ameliorates Demyelinating Phenotype in a Cellular and Animal Model of Charcot-Marie-Tooth Disease Type 1A

Charcot-Marie-Tooth disease (CMT) is a genetically heterogeneous disease affecting the peripheral nervous system that is caused by either the demyelination of Schwann cells or degeneration of the peripheral axon. Currently, there are no treatment options to improve the degeneration of peripheral nerves in CMT patients. In this research, we assessed the potency of farnesol for improving the demyelinating phenotype using an animal model of CMT type 1A. In vitro treatment with farnesol facilitated myelin gene expression and ameliorated the myelination defect caused by PMP22 overexpression, the major causative gene in CMT. In vivo administration of farnesol enhanced the peripheral neuropathic phenotype, as shown by rotarod performance in a mouse model of CMT1A. Electrophysiologically, farnesol-administered CMT1A mice exhibited increased motor nerve conduction velocity and compound muscle action potential compared with control mice. The number and diameter of myelinated axons were also increased by farnesol treatment. The expression level of myelin protein zero (MPZ) was increased, while that of the demyelination marker, neural cell adhesion molecule (NCAM), was reduced by farnesol administration. These data imply that farnesol is efficacious in ameliorating the demyelinating phenotype of CMT, and further elucidation of the underlying mechanisms of farnesol’s effect on myelination might provide a potent therapeutic strategy for the demyelinating type of CMT.     

Glia Disease and Repair—Remyelination

The inability of the mammalian central nervous system (CNS) to undergo spontaneous regeneration has long been regarded as a central tenet of neurobiology. However, although this is largely true of the neuronal elements of the adult mammalian CNS, save for discrete populations of granular neurons, the same is not true of its glial elements. In particular, the loss of oligodendrocytes, which results in demyelination, triggers a spontaneous and often highly efficient regenerative response, remyelination, in which new oligodendrocytes are generated and myelin sheaths are restored to denuded axons. Yet, remyelination in humans is not without limitation, and a variety of demyelinating conditions are associated with sustained and disabling myelin loss. In this review, we will review the biology of remyelination, including the cells and signals involved; describe when remyelination occurs and when and why it fails and the consequences of its failure; and discuss approaches for therapeutically enhancing remyelination in demyelinating diseases of both children and adults, both by stimulating endogenous oligodendrocyte progenitor cells and by transplanting these cells into demyelinated brain.     

Remyelination in the CNS: from biology to therapy

Remyelination involves reinvesting demyelinated axons with new myelin sheaths. In stark contrast to the situation that follows loss of neurons or axonal damage, remyelination in the CNS can be a highly effective regenerative process. It is mediated by a population of precursor cells called oligodendrocyte precursor cells (OPCs), which are widely distributed throughout the adult CNS. However, despite its efficiency in experimental models and in some clinical diseases, remyelination is often inadequate in demyelinating diseases such as multiple sclerosis (MS), the most common demyelinating disease and a cause of neurological disability in young adults. The failure of remyelination has profound consequences for the health of axons, the progressive and irreversible loss of which accounts for the progressive nature of these diseases. The mechanisms of remyelination therefore provide critical clues for regeneration biologists that help them to determine why remyelination fails in MS and in other demyelinating diseases and how it might be enhanced therapeutically.     

Optogenetic neuronal stimulation promotes axon outgrowth and myelination of motor neurons in a three-dimensional motor neuron–Schwann cell coculture model on a microfluidic biochip

Axonal regeneration and remyelination of peripheral motor neurons (MNs) are critical for restoring neuromuscular motor function after injury or peripheral neuropathy. We examined whether optogenetically mediated light stimulation (OMLS) could enhance the axon outgrowth and myelination of MNs using three-dimensional motor neuron–Schwann cell (MN–SC) coculture on a microfluidic biochip. The biochip was designed to allow SCs to interact with the axons of MNs, while preventing direct contact between SCs and the cell bodies of MNs. Following coculture with SCs on the microfluidic biochip, MNs were transfected with a light-sensitive channelrhodopsin gene. Transfected MNs subjected to repeated light stimulation (20 Hz, 1 hr) produced significantly longer axons than nontransfected MNs. OMLS of MNs greatly increased the number of myelin basic protein (MBP)-expressing SCs, promoting the initiation of myelination of MNs. Ultrastructurally, OMLS of MNs markedly enhanced the thickness of the compact myelin sheath around the MN axons such that the average thickness was closer to that of the theoretical estimates in vivo. Thus, the MN–SC coculture model on a microfluidic biochip augmented by OMLS of MNs is a feasible platform for studying the relationship of neuronal activity with regrowth and remyelination.     

Detection of sodium channel distribution in rat sciatic nerve following lysophosphatidylcholine-induced demyelination

Summary In vivo application of lysophosphatidylcholine (LPC) to rat sciatic nerve induces impaired hind leg movement within 2 days which is recovered by 6 days. Segmental demyelination was seen at 2 days after LPC application, and remyelination had barely started in a few axons by 6 days. Using sodium channel-specific monoclonal antibodies and immunofluorescence microscopy, we observed altered distribution of sodium channels in demyelinated axons. Bright fluorescent labeling was found along the segmentally demyelinated axolemma at 6 days in contrast to the dim staining of the demyelinated nerve found at 2 days. In addition, radioimmunoassays detected an elevated number of antibody binding sites on sciatic nerve trunk from the sixth day. Our data provide the immunocytochemical evidence for the assumption that recruitment of sodium channels into demyelinated axolemma contributes to the recovery of function following axon demyelination by LPC.     

Endogenous Lectin Cerebellar Soluble Lectin Involved in Myelination Is Absent from Nonmyelinating Schwann Cells

In the sciatic nerve, two major classes of Schwann cells are present which differ in their capability to produce myelin. Myelinating Schwann cells surround most of the axons with the formation of a typical myelin sheath. Nonmyelinating Schwann cells serve to insulate individual axons without formation of myelin. These dissimilarities between the two types of Schwann cells provided an interesting model for studying mechanisms underlying myelination and the formation of contacts between axons and myelinating cells. It is demonstrated here that the endogenous lectin cerebellar soluble lectin (CSL), implicated in myelin stabilization and in formation of contact between axon and myelinating cells in the CNS and in the sciatic nerve, is undetectable in non-myelinating Schwann cells. In contrast, most axons surrounded by these cells contained the major axonal glycoprotein ligand of CSL, a 31-kDa glycoprotein which is present in large amounts. The possible relationship between the presence of CSL in Schwann cells and their capacity to interact with axons and to produce myelin are discussed.     

Oxydative phosphorylation in sciatic nerve myelin and its impairment in a model of dysmyelinating peripheral neuropathy

Myelin sheath is the proteolipid membrane wrapping the axons of CNS and PNS. We have shown data suggesting that CNS myelin conducts oxidative phosphorylation (OXPHOS), challenging its role in limiting the axonal energy expenditure. Here, we focused on PNS myelin. Samples were: (i) isolated myelin vesicles (IMV) from sciatic nerves, (ii) mitochondria from primary Schwann cell cultures, and (iii) sciatic nerve sections, from wild type or Charcot‐Marie‐Tooth type 1A (CMT1A) rats. The latter used as a model of dys‐demyelination. O₂ consumption and activity of OXPHOS proteins from wild type (Wt) or CMT1A sciatic nerves showed some differences. In particular, O₂ consumption by IMV from Wt and CMT1A 1‐month‐old rats was comparable, while it was severely impaired in IMV from adult affected animals. Mitochondria extracted from CMT1A Schwann cell did not show any dysfunction. Transmission electron microscopy studies demonstrated an increased mitochondrial density in dys‐demyelinated axons, as to compensate for the loss of respiration by myelin. Confocal immunohistochemistry showed the expression of OXPHOS proteins in the myelin sheath, both in Wt and dys‐demyelinated nerves. These revealed an abnormal morphology. Taken together these results support the idea that also PNS myelin conducts OXPHOS to sustain axonal function.     

Schwann cell autophagy,myelinophagy, initiates myelin clearance from injured nerves

Although Schwann cell myelin breakdown is the universal outcome of a remarkably wide range of conditions that cause disease or injury to peripheral nerves, the cellular and molecular mechanisms that make Schwann cell–mediated myelin digestion possible have not been established. We report that Schwann cells degrade myelin after injury by a novel form of selective autophagy, myelinophagy. Autophagy was up-regulated by myelinating Schwann cells after nerve injury, myelin debris was present in autophagosomes, and pharmacological and genetic inhibition of autophagy impaired myelin clearance. Myelinophagy was positively regulated by the Schwann cell JNK/c-Jun pathway, a central regulator of the Schwann cell reprogramming induced by nerve injury. We also present evidence that myelinophagy is defective in the injured central nervous system. These results reveal an important role for inductive autophagy during Wallerian degeneration, and point to potential mechanistic targets for accelerating myelin clearance and improving demyelinating disease.     

Myelination and node of Ranvier formation on sensory neurons in a defined in vitro system

One of the most important developmental modifications of the nervous system is Schwann cell myelination of axons. Schwann cells ensheath axons to create myelin segments to provide protection to the axon as well as increase the conduction of action potentials. In vitro neuronal systems provide a unique modality to study a variety of factors influencing myelination as well as diseases associated with myelin sheath degradation. This work details the development of a patterned in vitro myelinating dorsal root ganglion culture. This defined system utilized a serum-free medium in combination with a patterned substrate, utilizing the cytophobic and cytophilic molecules (poly)ethylene glycol (PEG) and N-1[3 (trimethoxysilyl) propyl] diethylenetriamine (DETA), respectively. Directional outgrowth of the neurites and subsequent myelination was controlled by surface modifications, and conformity to the pattern was measured over the duration of the experiments. The myelinated segments and nodal proteins were visualized and quantified using confocal microscopy. This tissue-engineered system provides a highly controlled, reproducible model for studying Schwann cell interactions with sensory neurons, as well as the myelination process, and its effect on neuronal plasticity and peripheral nerve regeneration. It is also compatible for use in bio-hybrid constructs to reproduce the stretch reflex arc on a chip because the media combination used is the same that we have used previously for motoneurons, muscle, and for neuromuscular junction (NMJ) formation. This work could have application for the study of demyelinating diseases such as diabetes induced peripheral neuropathy and could rapidly translate to a role in the discovery of drugs promoting enhanced peripheral nervous system (PNS) remyelination.