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Background: Traumatic brain injury (TBI) causes axon tearing and synapse degradation, resulting in multiple neurological dysfunctions and exacerbation of early neurodegeneration; the repair of axonal and synaptic structures is critical for restoring neuronal function. C-C Motif Chemokine Ligand 5 (CCL5) shows many neuroprotective activities., Method: A close-head weight-drop system was used to induce mild brain trauma in C57BL/6 (wild-type, WT) and CCL5 knockout (CCL5-KO) mice. The mNSS score, rotarod, beam walking, and sticker removal tests were used to assay neurological function after mTBI in different groups of mice. The restoration of motor and sensory functions was impaired in CCL5-KO mice after one month of injury, with swelling of axons and synapses from Golgi staining and reduced synaptic proteins-synaptophysin and PSD95. Administration of recombinant CCL5 (Pre-treatment: 300 pg/g once before injury; or post-treatment: 30 pg/g every 2 days, since 3 days after injury for 1 month) through intranasal delivery into mouse brain improved the motor and sensory neurological dysfunctions in CCL5-KO TBI mice., Results: Proteomic analysis using LC-MS/MS identified that the "Nervous system development and function"-related proteins, including axonogenesis, synaptogenesis, and myelination signaling pathways, were reduced in injured cortex of CCL5-KO mice; both pre-treatment and post-treatment with CCL5 augmented those pathways. Immunostaining and western blot analysis confirmed axonogenesis and synaptogenesis related Semaphorin, Ephrin, p70S6/mTOR signaling, and myelination-related Neuregulin/ErbB and FGF/FAK signaling pathways were up-regulated in the cortical tissue by CCL5 after brain injury. We also noticed cortex redevelopment after long-term administration of CCL5 after brain injury with increased Reelin positive Cajal-Rerzius Cells and CXCR4 expression. CCL5 enhanced the growth of cone filopodia in a primary neuron culture system; blocking CCL5's receptor CCR5 by Maraviroc reduced the intensity of filopodia in growth cone and also CCL5 mediated mTOR and Rho signalling activation. Inhibiting mTOR and Rho signaling abolished CCL5 induced growth cone formation., Conclusions: CCL5 plays a critical role in starting the intrinsic neuronal regeneration system following TBI, which includes growth cone formation, axonogenesis and synaptogensis, remyelination, and the subsequent proper wiring of cortical circuits. Our study underscores the potential of CCL5 as a robust therapeutic stratagem in treating axonal injury and degeneration during the chronic phase after mild brain injury., (© 2024. The Author(s).)

Traumatic brain injury (TBI) has a significant impact on cognitive function, affecting millions of people worldwide. Myelin loss is a prominent pathological feature of TBI, while well-functioning myelin is crucial for memory and cognition. Utilizing drug repurposing to identify effective drug candidates for TBI treatment has gained attention. Notably, recent research has highlighted the potential of clemastine, an FDA-approved allergy medication, as a promising pro-myelinating drug. Therefore, in this study, we aim to investigate whether clemastine can enhance myelination and alleviate cognitive impairment following mild TBI using a clinically relevant rat model of TBI. Mild diffuse TBI was induced using the Closed-Head Impact Model of Engineered Rotational Acceleration (CHIMERA). Animals were treated with either clemastine or an equivalent volume of the vehicle from day 1 to day 14 post-injury. Following treatment, memory-related behavioral tests were conducted, and myelin pathology in the cortex and hippocampus was assessed through immunofluorescence staining and ProteinSimple® capillary-based immunoassay. Our results showed that TBI leads to significant myelin loss, axonal damage, glial activation, and a decrease in mature oligodendrocytes in both the cortex and hippocampus. The TBI animals also exhibited notable deficits in memory-related tests. In contrast, animals treated with clemastine showed an increase in mature oligodendrocytes, enhanced myelination, and improved performance in the behavioral tests. These preliminary findings support the therapeutic value of clemastine in alleviating TBI-induced cognitive impairment, with substantial clinical translational potential. Our findings also underscore the potential of remyelinating therapies for TBI., Competing Interests: Declaration of competing interest None., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Axons of globular bushy cells in the cochlear nucleus convey hyper-accurate signals to the superior olivary complex, the initial site of binaural processing via comparably thick axons and the calyx of the Held synapse. Bushy cell fibers involved in hyper-accurate binaural processing of low-frequency sounds are known to have an unusual internode length-to-axon caliber ratio (L/d) correlating with higher conduction velocity and superior temporal precision of action potentials. How the L/d-ratio develops and what determines this unusual myelination pattern is unclear. Here we describe a gradual developmental transition from very simple to complex, mature nodes of Ranvier on globular bushy cell axons during a 2-week period starting at postnatal day P6/7. The molecular composition of nodes matured successively along the axons from somata to synaptic terminals with morphologically and molecularly mature nodes appearing almost exclusively after hearing onset. Internodal distances are initially coherent with the canonical L/d-ratio of ~100. Several days after hearing onset, however, an over-proportional increase in axon caliber occurs in cells signaling low-frequency sounds which alters their L/d ratio to ~60. Hence, oligodendrocytes initially myelinating axons according to their transient axon caliber but a subsequent differential axon thickening after hearing onset results in the unusual myelination pattern., (© 2024 The Authors. GLIA published by Wiley Periodicals LLC.)

Adaptive myelination is the emerging concept of tuning axonal conduction velocity to the activity within specific neural circuits over time. Sound processing circuits exhibit structural and functional specifications to process signals with microsecond precision: a time scale that is amenable to adjustment in length and thickness of myelin. Increasing activity of auditory axons by introducing sound-evoked responses during postnatal development enhances myelin thickness, while sensory deprivation prevents such radial growth during development. When deprivation occurs during adulthood, myelin thickness was reduced. However, it is unclear whether sensory stimulation adjusts myelination in a global fashion (whole fiber bundles) or whether such adaptation occurs at the level of individual fibers. Using temporary monaural deprivation in mice provided an internal control for a) differentially tracing structural changes in active and deprived fibers and b) for monitoring neural activity in response to acoustic stimulation of the control and the deprived ear within the same animal. The data show that sound-evoked activity increased the number of myelin layers around individual active axons, even when located in mixed bundles of active and deprived fibers. Thicker myelination correlated with faster axonal conduction velocity and caused shorter auditory brainstem response wave VI-I delays, providing a physiologically relevant readout. The lack of global compensation emphasizes the importance of balanced sensory experience in both ears throughout the lifespan of an individual., Competing Interests: Competing interests statement:The authors declare no competing interest.

Axons undergo striking changes in their content and distribution of cell adhesion molecules (CAMs) and ion channels during myelination that underlies the switch from continuous to saltatory conduction. These changes include the removal of a large cohort of uniformly distributed CAMs that mediate initial axon-Schwann cell interactions and their replacement by a subset of CAMs that mediate domain-specific interactions of myelinated fibers. Here, using rodent models, we examine the mechanisms and significance of this removal of axonal CAMs. We show that Schwann cells just prior to myelination locally activate clathrin-mediated endocytosis (CME) in axons, thereby driving clearance of a broad array of axonal CAMs. CAMs engineered to resist endocytosis are persistently expressed along the axon and delay both PNS and CNS myelination. Thus, glia non-autonomously activate CME in axons to downregulate axonal CAMs and presumptively axo-glial adhesion. This promotes the transition from ensheathment to myelination while simultaneously sculpting the formation of axonal domains., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Oligodendrocytes in the central nervous system exhibit significant variability in the number of myelin sheaths that are supported by each cell, ranging from 1 to 50 (1-8). Myelin production during development is dynamic and involves both sheath formation and loss (3, 9-13). However, how these parameters are balanced to generate this heterogeneity in sheath number has not been thoroughly investigated. To explore this question, we combined extensive time-lapse and longitudinal imaging of oligodendrocytes in the developing zebrafish spinal cord to quantify sheath initiation and loss. Surprisingly, we found that oligodendrocytes repetitively ensheathed the same axons multiple times before any stable sheaths were formed. Importantly, this repetitive ensheathment was independent of neuronal activity. At the level of individual oligodendrocytes, each cell initiated a highly variable number of total ensheathments. However, ~80-90% of these ensheathments always disappeared, an unexpectedly high, but consistent rate of loss. The dynamics of this process indicated rapid membrane turnover as ensheathments were formed and lost repetitively on each axon. To better understand how these sheath initiation dynamics contribute to sheath accumulation and stabilization, we disrupted membrane recycling by expressing a dominant-negative mutant form of Rab5. Oligodendrocytes over-expressing this mutant did not show a change in early sheath initiation dynamics but did lose a higher percentage of ensheathments in the later stabilization phase. Overall, oligodendrocyte sheath number is heterogeneous because each cell repetitively initiates a variable number of total ensheathments that are resolved through a consistent stabilization rate., Competing Interests: AA, WM No competing interests declared, (© 2023, Almeida and Macklin.)

During development, dramatic changes in myelination, growth of neural networks and changes in grey-to-white matter ratio build up the astonishingly plastic brain of a child. The progressive increase in myelination insulates the nervous system, which, in turn, modifies the mechanical microenvironment of the brain spatiotemporally. A growing body of evidence demonstrates the role of mechanical forces in growth, differentiation, maturation and electrical properties of neurons. However, due to limitations in imaging resolution, the exact relationship between myelination, axonal organization and the mechanical properties of nerves at the cellular level is still unknown. Here, we propose a novel approach to study the direct relationship between axonal viscoelasticity with changing fibre anisotropy and myelination during development. With the use of atomic force microscopy (AFM) with in situ fluorescent imaging of the primary neuron-oligodendrocyte co-cultures, we found that as axons are progressively myelinated in vitro, their stiffness increases. Direct quantification of myelin along axons using immunofluorescence also demonstrated a positive correlation between increased myelination over time and increased axonal stiffness (p = .001). Notably, AFM measurements along a single axon showed that the Young's modulus measured across myelinated regions were significantly higher than those of adjacent unmyelinated segments at all time points (p < .0001). Force-relaxation analysis also demonstrated that myelin sheath dominates the regulation of viscoelasticity of axons temporally. Collectively, our findings indicate a direct link between myelination, axonal orientation and viscoelasticity, providing important insights about the mechanical environment in the paediatric brain, with direct implications for our understanding of developmental brain disorders and paediatric brain injury., (© 2023 Federation of European Neuroscience Societies and John Wiley & Sons Ltd.)

Synchronization of network oscillation in spatially distant cortical areas is essential for normal brain activity. Precision in synchronization between hemispheres depends on the axonal conduction velocity, which is determined by physical parameters of the axons involved, including diameter, and extent of myelination. To compare these parameters in long-projecting excitatory and inhibitory axons in the corpus callosum, we used genetically modified mice and virus tracing to separately label CaMKIIα expressing excitatory and GABAergic inhibitory axons. Using electron microscopy analysis, we revealed that (i) the axon diameters of excitatory fibers (myelinated axons) are significantly larger than those of nonmyelinated excitatory axons; (ii) the diameters of bare axons of excitatory myelinated fibers are significantly larger than those of their inhibitory counterparts; and (iii) myelinated excitatory fibers are significantly larger than myelinated inhibitory fibers. Also, the thickness of myelin ensheathing inhibitory axons is significantly greater than for excitatory axons, with the ultrastructure of the myelin around excitatory and inhibitory fibers also differing. We generated a computational model to investigate the functional consequences of these parameter divergences. Our simulations indicate that impulses through inhibitory and excitatory myelinated fibers reach the target almost simultaneously, whereas action potentials conducted by nonmyelinated axons reach target cells with considerable delay., (© The Author(s) 2022. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permission@oup.com.)

Oligodendrocyte production and myelination continues lifelong in the central nervous system (CNS), and all stages of this process can be adaptively regulated by neuronal activity. While artificial exogenous stimulation of neuronal circuits greatly enhances oligodendrocyte progenitor cell (OPC) production and increases myelination during development, the extent to which physiological stimuli replicates this is unclear, particularly in the adult CNS when the rate of new myelin addition slows. Here, we used environmental enrichment (EE) to physiologically stimulate neuronal activity for 6 weeks in 9-week-old C57BL/six male and female mice and found no increase in compact myelin in the corpus callosum or somatosensory cortex. Instead, we observed a global increase in callosal axon diameter with thicker myelin sheaths, elongated paranodes and shortened nodes of Ranvier. These findings indicate that EE induced the dynamic structural remodelling of myelinated axons. Additionally, we observed a global increase in the differentiation of OPCs and pre-myelinating oligodendroglia in the corpus callosum and somatosensory cortex. Our findings of structural remodelling of myelinated axons in response to physiological neural stimuli during young adulthood provide important insights in understanding experience-dependent myelin plasticity throughout the lifespan and provide a platform to investigate axon-myelin interactions in a physiologically relevant context., (© 2022 The Authors. European Journal of Neuroscience published by Federation of European Neuroscience Societies and John Wiley & Sons Ltd.)

The spacing of nodes of Ranvier crucially affects conduction properties along myelinated axons. It is assumed that node position is primarily driven by growing myelin sheaths. Here, we reveal an additional mechanism of node positioning that is driven by the axon. Through longitudinal live imaging of node formation dynamics in the zebrafish central nervous system, we show that stable clusters of the cell adhesion molecule neurofascin a can accumulate at specific sites along axons prior to myelination. While some of these clusters are pushed into future node position by extending myelin sheaths, others are not and thus prefigure the position of where a mature node forms. Animals that lack full-length neurofascin a show increased internodal distances and less regular nodal spacing along single axons. Together, our data reveal the existence of an axonal mechanism to position nodes of Ranvier that does not depend on regulation by myelin sheath growth., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Axonal conduction velocity, which ensures efficient function of the brain network, is related to axon diameter. Noninvasive, in vivo axon diameter estimates can be made with diffusion magnetic resonance imaging, but the technique requires three-dimensional (3D) validation. Here, high-resolution, 3D synchrotron X-ray nano-holotomography images of white matter samples from the corpus callosum of a monkey brain reveal that blood vessels, cells, and vacuoles affect axonal diameter and trajectory. Within single axons, we find that the variation in diameter and conduction velocity correlates with the mean diameter, contesting the value of precise diameter determination in larger axons. These complex 3D axon morphologies drive previously reported 2D trends in axon diameter and g -ratio. Furthermore, we find that these morphologies bias the estimates of axon diameter with diffusion magnetic resonance imaging and, ultimately, impact the investigation and formulation of the axon structure-function relationship., Competing Interests: The authors declare no competing interest., (Copyright © 2020 the Author(s). Published by PNAS.)

Myelination facilitates rapid axonal conduction, enabling efficient communication across different parts of the nervous system. Here we examined mechanisms controlling myelination after injury and during axon regeneration in the central nervous system (CNS). Previously, we discovered multiple molecular pathways and strategies that could promote robust axon regrowth after optic nerve injury. However, regenerated axons remain unmyelinated, and the underlying mechanisms are elusive. In this study, we found that, in injured optic nerves, oligodendrocyte precursor cells (OPCs) undergo transient proliferation but fail to differentiate into mature myelination-competent oligodendrocytes, reminiscent of what is observed in human progressive multiple sclerosis. Mechanistically, we showed that OPC-intrinsic GPR17 signaling and sustained activation of microglia inhibit different stages of OPC differentiation. Importantly, co-manipulation of GPR17 and microglia led to extensive myelination of regenerated axons. The regulatory mechanisms of stage-dependent OPC differentiation uncovered here suggest a translatable strategy for efficient de novo myelination after CNS injury., Competing Interests: Declaration of Interests A patent based on the results in this manuscript was filed by Boston Children’s Hospital (Z.H., J.W., and X.H. are co-inventors)., (Copyright © 2020 Elsevier Inc. All rights reserved.)

In the developing spinal cord, the majority of oligodendrocyte progenitor cells (OPCs) are induced in the ventral neuroepithelium under the control of the Sonic Hedgehog (Shh) signaling pathway, whereas a small subset of OPCs are generated from the dorsal neuroepithelial cells independent of the Shh pathway. Although dorsally-derived OPCs (dOPCs) have been shown to participate in local axonal myelination in the dorsolateral regions during development, it is not known whether they are capable of migrating into the ventral region and myelinating ventral axons. In this study, we confirmed and extended the previous study on the developmental potential of dOPCs in the absence of ventrally-derived OPCs (vOPCs). In Nestin-Smo conditional knockout (cKO) mice, when ventral oligodendrogenesis was blocked, dOPCs were found to undergo rapid amplification, spread to ventral spinal tissue, and eventually differentiated into myelinating OLs in the ventral white matter with a temporal delay, providing genetic evidence that dOPCs are capable of myelinating ventral axons in the mouse spinal cord.

Background: In the developing central nervous system, pre-myelinating oligodendrocytes sample candidate nerve axons by extending and retracting process extensions. Some contacts stabilize, leading to the initiation of axon wrapping, nascent myelin sheath formation, concentric wrapping and sheath elongation, and sheath stabilization or pruning by oligodendrocytes. Although axonal signals influence the overall process of myelination, the precise oligodendrocyte behaviors that require signaling from axons are not completely understood. In this study, we investigated whether oligodendrocyte behaviors during the early events of myelination are mediated by an oligodendrocyte-intrinsic myelination program or are over-ridden by axonal factors., Methods: To address this, we utilized in vivo time-lapse imaging in embryonic and larval zebrafish spinal cord during the initial hours and days of axon wrapping and myelination. Transgenic reporter lines marked individual axon subtypes or oligodendrocyte membranes., Results: In the larval zebrafish spinal cord, individual axon subtypes supported distinct nascent sheath growth rates and stabilization frequencies. Oligodendrocytes ensheathed individual axon subtypes at different rates during a two-day period after initial axon wrapping. When descending reticulospinal axons were ablated, local spinal axons supported a constant ensheathment rate despite the increased ratio of oligodendrocytes to target axons., Conclusion: We conclude that properties of individual axon subtypes instruct oligodendrocyte behaviors during initial stages of myelination by differentially controlling nascent sheath growth and stabilization.

In vertebrates, fast saltatory conduction along myelinated axons relies on the node of Ranvier. How nodes assemble on CNS neurons is not yet fully understood. We previously described that node-like clusters can form prior to myelin deposition in hippocampal GABAergic neurons and are associated with increased conduction velocity. Here, we used a live imaging approach to characterize the intrinsic mechanisms underlying the assembly of these clusters prior to myelination. We first demonstrated that their components can partially preassemble prior to membrane targeting and determined the molecular motors involved in their trafficking. We then demonstrated the key role of the protein β2Na v for node-like clustering initiation. We further assessed the fate of these clusters when myelination proceeds. Our results shed light on the intrinsic mechanisms involved in node-like clustering prior to myelination and unravel a potential role of these clusters in node of Ranvier formation and in guiding myelination onset., (© 2020 Wiley Periodicals, Inc.)

Complex signaling between Schwann cells and axons are vital for peripheral neuron development, myelination, and repair. The interaction between these two cell types can be modeled in vitro by coculturing rodent Schwann cells and neurons together. These have in the past been used with great success to help unravel the bidirectional signaling mechanisms that lead to Schwann cell proliferation and myelination. To provide more translatable potential, we have developed myelinating cocultures using human, induced pluripotent stem cell (iPSC)-derived neurons. Under the right conditions, the human neurons are efficiently myelinated by rat Schwann cells, demonstrating successful cross-species signaling. This chapter describes all the necessary steps to generate these myelinating cocultures and methods to investigate and quantify various aspects of myelination. The myelinating cocultures can be maintained in excellent health for over 1 year, facilitating their use to study developmental or chronic disease processes. With this in mind, we have used the cocultures to model a sensory neuropathy which displays clinically with both axonal and demyelinating features. In the cocultures, we found evidence of extensive axonal degeneration and demyelination demonstrated by axonal swelling and fragmentation, and myelin disintegration. The myelinating cocultures can therefore be used to study complex, human disease processes that result in both axonal and myelin-associated degenerative processes.

The paper presents a hierarchical series of computational models for myelinated axonal compartments. Three classes of models are considered, either with distributed parameters (2.5D EQS-ElectroQuasi Static, 1D TL-Transmission Lines) or with lumped parameters (0D). They are systematically analyzed with both analytical and numerical approaches, the main goal being to identify the best procedure for order reduction of each case. An appropriate error estimator is proposed in order to assess the accuracy of the models. This is the foundation of a procedure able to find the simplest reduced model having an imposed precision. The most computationally efficient model from the three geometries proved to be the analytical 1D one, which is able to have accuracy less than 0.1%. By order reduction with vector fitting, a finite model is generated with a relative difference of 10 - 4 for order 5. The dynamical models thus extracted allow an efficient simulation of neurons and, consequently, of neuronal circuits. In such situations, the linear models of the myelinated compartments coupled with the dynamical, non-linear models of the Ranvier nodes, neuronal body (soma) and dendritic tree give global reduced models. In order to ease the simulation of large-scale neuronal systems, the sub-models at each level, including those of myelinated compartments should have the lowest possible order. The presented procedure is a first step in achieving simulations of neural systems with accuracy control.

GABAergic fast-spiking parvalbumin-positive (PV) interneurons are frequently myelinated in the cerebral cortex. However, the factors governing the topography of cortical interneuron myelination remain incompletely understood. Here, we report that segmental myelination along neocortical interneuron axons is strongly predicted by the joint combination of interbranch distance and local axon caliber. Enlargement of PV+ interneurons increased axonal myelination, while reduced cell size led to decreased myelination. Next, we considered regular-spiking SOM+ cells, which normally have relatively shorter interbranch distances and thinner axon diameters than PV+ cells, and are rarely myelinated. Consistent with the importance of axonal morphology for guiding interneuron myelination, enlargement of SOM+ cell size dramatically increased the frequency of myelinated axonal segments. Lastly, we confirm that these findings also extend to human neocortex by quantifying interneuron axonal myelination from ex vivo surgical tissue. Together, these findings establish a predictive model of neocortical GABAergic interneuron myelination determined by local axonal morphology., Competing Interests: JS, DB, JS, MP, ML, BB, CD, ZG, DB, AH, SK No competing interests declared, (© 2019, Stedehouder et al.)

Diabetes mellitus-induced hyperglycemia is associated with a number of pathologies such as retinopathy, nephropathy, delayed wound healing, and diabetic peripheral neuropathy (DPN). Approximately 50% of patients with diabetes mellitus will develop DPN, which is characterized by disrupted sensory and/or motor functioning, with treatment limited to pain management. Zebrafish ( Danio rerio ) are an emerging animal model used to study a number of metabolic disorders, including diabetes. Diabetic retinopathy, nephropathy, and delayed wound healing have all been demonstrated in zebrafish. Recently, our laboratory has demonstrated that following the ablation of the insulin-producing β-cells of the pancreas (and subsequent hyperglycemia), the peripheral nerves begin to show signs of dysregulation. In this study, we take a different approach, taking advantage of the transdermal absorption abilities of zebrafish larvae to extend the period of hyperglycemia. Following 5 days of 60 mM d-glucose treatment, we observed motor axon defasciculation, disturbances in perineurial glia sheath formation, decreased myelination of motor axons, and sensory neuron mislocalization. This study extends our understanding of the structural changes of the peripheral nerve following induction of hyperglycemia and does so in an animal model capable of potential DPN drug discovery in the future. NEW & NOTEWORTHY Zebrafish are emerging as a robust model system for the study of diabetic complications such as retinopathy, nephropathy, and impaired wound healing. We present a novel model of diabetic peripheral neuropathy in zebrafish in which the integrity of the peripheral nerve is dysregulated following the induction of hyperglycemia. By using this model, future studies can focus on elucidating the underlying molecular mechanisms currently unknown.

Reciprocal communication between neurons and oligodendrocytes is essential for the generation and localization of myelin, a critical feature of the CNS. In the neocortex, individual oligodendrocytes can myelinate multiple axons; however, the neuronal origin of the myelinated axons has remained undefined and, while largely assumed to be from excitatory pyramidal neurons, it also includes inhibitory interneurons. This raises the question of whether individual oligodendrocytes display bias for the class of neurons that they myelinate. Here, we find that different classes of cortical interneurons show distinct patterns of myelin distribution starting from the onset of myelination, suggesting that oligodendrocytes can recognize the class identity of individual types of interneurons that they target. Notably, we show that some oligodendrocytes disproportionately myelinate the axons of inhibitory interneurons, whereas others primarily target excitatory axons or show no bias. These results point toward very specific interactions between oligodendrocytes and neurons and raise the interesting question of why myelination is differentially directed toward different neuron types., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

Nodes of Ranvier are unique regions where voltage-gated sodium channels are highly enriched to drive saltatory conduction. Genetic ablations in adult mice with loss of specific nodal proteins causes slow but progressive nodal deterioration associated with decreased nerve conduction and axonopathy. What has remained unaddressed is whether loss of nodal proteins at different time points in postnatal life follows similar timelines of nodal disorganization. Here we utilized simultaneous ablation of Neurofascin (NF186) and Ankyrin G (AnkG) in mice of both sexes at three specific time points. We report that concurrent ablation of these core nodal components at postnatal day 13 (P13) leads to accelerated nodal destabilization in comparison with P23, and this disorganization is even slower when ablated at P93. Ablation of NF186 with AnkG at P13 reduced the half-life of NF186 to 15 days compared to 1 month at P23, which increased to 2 months at P93, indicating increasing nodal stability. The half-life of AnkG at the nodes also increased with age but showed enhanced disappearance from the node in the absence of NF186, with a half-life of 3 days at P13 ablation. The nodal disorganization occurred in a sequential manner, with AnkG disappearing first from the nodal areas irrespective of the timing of ablation, and led to decreased nerve conduction and affected axonal health. Together, our studies reveal that nodes of Ranvier in myelinated axons continue to become more stable with age and suggest that nodal disorganization in adult human demyelinating disorders occurs slowly until neurological symptoms become evident.

Background: Myelination, degeneration and regeneration are implicated in crucial responses to injury in the peripheral nervous system. Considering the progression of amyotrophic lateral sclerosis (ALS), we used the superoxide dismutase 1 (SOD1)-G93A transgenic mouse model of ALS to investigate the effects of mutant SOD1 on the peripheral nerves., Methods: Changes in peripheral nerve morphology were analyzed in SOD1 mutant mice at various stages of the disease by toluidine blue staining and electron microscopy (EM). Schwann cell proliferation and recruitment of inflammatory factors were detected by immunofluorescence staining and quantitative reverse transcription PCR and were compared between SOD1 mutant mice and control mice. Furthermore, western blotting (WB) and TUNEL staining were used to investigate axonal damage and Schwann cell survival in the sciatic nerves of mice in both groups., Results: An analysis of the peripheral nervous system in SOD1-G93A mice revealed the following novel features: (i) Schwann cells and axons in mutant mice underwent changes that were similar to those seen in the control mice during the early development of peripheral nerves. (ii) The peripheral nerves of SOD1-G93A mice developed progressive neuropathy, which presented as defects in axons and myelin, leading to difficulty in walking and reduced locomotor capacity at a late stage of the disease. (iii) Macrophages were recruited and accumulated, and nerve injury and a deficit in the blood-nerve barrier were observed. (iv) Proliferation and the inflammatory micro-environment were inhibited, which impaired the regeneration and remyelination of axons after crush injury in the SOD1-G93A mice., Conclusions: The mutant human SOD1 protein induced axonal and myelin degeneration during the progression of ALS and participated in axon remyelination and regeneration in response to injury., (© 2018 The Author(s). Published by S. Karger AG, Basel.)

The formation of new myelin in persistent multiple sclerosis (MS) lesions is compromised, leading to a reduction in neuron function and subject to degeneration and death. Current MS therapies can control autoimmune-mediated demyelination, but none directly promote the regeneration of myelin in the central nervous system (CNS). To identify new drugs that stimulate remyelination, we established a high-throughput cell-based assay to identify compounds that promote myelination. Methods were developed for initiating myelination in vitro using a preparation of primary embryonic rat cortical cells. We developed an immunofluorescent phenotypic image analysis method to quantify the morphological alignment of myelin characteristic of the initiation of myelination. The assay scalability and consistency was validated by screening the NIH clinical collection library of 727 compounds and identified ten compounds that promote myelination (Lariosa-Willingham et al., BMC Neurosci 17:16, 2016). Here, we present the detailed methods for a high capacity in vitro assay that assesses myelination of live axons.

Plasticity of myelination represents a mechanism to tune the flow of information by balancing functional requirements with metabolic and spatial constraints. The auditory system is heavily myelinated and operates at the upper limits of action potential generation frequency and speed observed in the mammalian CNS. This study aimed to characterize the development of myelin within the trapezoid body, a central auditory fiber tract, and determine the influence sensory experience has on this process in mice of both sexes. We find that in vitro conduction speed doubles following hearing onset and the ability to support high-frequency firing increases concurrently. Also in this time, the diameter of trapezoid body axons and the thickness of myelin double, reaching mature-like thickness between 25 and 35 d of age. Earplugs were used to induce ∼50 dB elevation in auditory thresholds. If introduced at hearing onset, trapezoid body fibers developed thinner axons and myelin than age-matched controls. If plugged during adulthood, the thickest trapezoid body fibers also showed a decrease in myelin. These data demonstrate the need for sensory activity in both development and maintenance of myelin and have important implications in the study of myelin plasticity and how this could relate to sensorineural hearing loss following peripheral impairment. SIGNIFICANCE STATEMENT The auditory system has many mechanisms to maximize the dynamic range of its afferent fibers, which operate at the physiological limit of action potential generation, precision, and speed. In this study we demonstrate for the first time that changes in peripheral activity modifies the thickness of myelin in sensory neurons, not only in development but also in mature animals. The current study suggests that changes in CNS myelination occur as a downstream mechanism following peripheral deficit. Given the required submillisecond temporal precision for binaural auditory processing, reduced myelination might augment sensorineural hearing impairment., (Copyright © 2017 Sinclair et al.)

This review article focuses on the Contactin (CNTN) subset of the Immunoglobulin supergene family (IgC2/FNIII molecules), whose components share structural properties (the association of Immunoglobulin type C2 with Fibronectin type III domains), as well as a general role in cell contact formation and axonal growth control. IgC2/FNIII molecules include 6 highly related components (CNTN 1-6), associated with the cell membrane via a Glycosyl Phosphatidyl Inositol (GPI)-containing lipid tail. Contactin 1 and Contactin 2 share ~50 (49.38)% identity at the aminoacid level. They are components of the cell surface, from which they may be released in soluble forms. They bind heterophilically to multiple partners in cis and in trans, including members of the related L1CAM family and of the Neurexin family Contactin-associated proteins (CNTNAPs or Casprs). Such interactions are important for organising the neuronal membrane, as well as for modulating the growth and pathfinding of axon tracts. In addition, they also mediate the functional maturation of axons by promoting their interactions with myelinating cells at the nodal, paranodal and juxtaparanodal regions. Such interactions also mediate differential ionic channels (both Na + and K + ) distribution, which is of critical relevance in the generation of the peak-shaped action potential. Indeed, thanks to their interactions with Ankyrin G, Na + channels map within the nodal regions, where they drive axonal depolarization. However, no ionic channels are found in the flanking Contactin1-containing paranodal regions, where CNTN1 interactions with Caspr1 and with the Ig superfamily component Neurofascin 155 in cis and in trans, respectively, build a molecular barrier between the node and the juxtaparanode. In this region K + channels are clustered, depending upon molecular interactions with Contactin 2 and with Caspr2. In addition to these functions, the Contactins appear to have also a role in degenerative and inflammatory disorders: indeed Contactin 2 is involved in neurodegenerative disorders with a special reference to the Alzheimer disease, given its ability to work as a ligand of the Alzheimer Precursor Protein (APP), which results in increased Alzheimer Intracellular Domain (AICD) release in a γ-secretase-dependent manner. On the other hand Contactin 1 drives Notch signalling activation via the Hes pathway, which could be consistent with its ability to modulate neuroinflammation events, and with the possibility that Contactin 1-dependent interactions may participate to the pathogenesis of the Multiple Sclerosis and of other inflammatory disorders., (Copyright © 2016 Elsevier Inc. All rights reserved.)

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., (© 2017 Wiley Periodicals, Inc.)

Adjusting the thickness and internodal length of the myelin sheath is a mechanism for tuning the conduction velocity of axons to match computational needs. Interactions between oligodendrocyte precursor cells (OPCs) and developing axons regulate the formation of myelin around axons. We now show, using organotypic cerebral cortex slices from mice expressing eGFP in Sox10-positive oligodendrocytes, that endogenously released GABA, acting on GABA A receptors, greatly reduces the number of oligodendrocyte lineage cells. The decrease in oligodendrocyte number correlates with a reduction in the amount of myelination but also an increase in internode length, a parameter previously thought to be set by the axon diameter or to be a property intrinsic to oligodendrocytes. Importantly, while TTX block of neuronal activity had no effect on oligodendrocyte lineage cell number when applied alone, it was able to completely abolish the effect of blocking GABA A receptors, suggesting that control of myelination by endogenous GABA may require a permissive factor to be released from axons. In contrast, block of AMPA/KA receptors had no effect on oligodendrocyte lineage cell number or myelination. These results imply that, during development, GABA can act as a local environmental cue to control myelination and thus influence the conduction velocity of action potentials within the CNS. GLIA 2017;65:309-321., (© 2016 The Authors Glia Published by Wiley Periodicals, Inc.)

Myelin is a lipid-rich sheath formed by the spiral wrapping of specialized glial cells around axon segments. Myelinating glia allow for rapid transmission of nerve impulses and metabolic support of axons, and the absence of or disruption to myelin results in debilitating motor, cognitive, and emotional deficits in humans. Because myelin is a jawed vertebrate innovation, zebrafish are one of the simplest vertebrate model systems to study the genetics and development of myelinating glia. The morphogenetic cellular movements and genetic program that drive myelination are conserved between zebrafish and mammals, and myelin develops rapidly in zebrafish larvae, within 3-5days postfertilization. Myelin ultrastructure can be visualized in the zebrafish from larval to adult stages via transmission electron microscopy, and the dynamic development of myelinating glial cells may be observed in vivo via transgenic reporter lines in zebrafish larvae. Zebrafish are amenable to genetic and pharmacological screens, and screens for myelinating glial phenotypes have revealed both genes and drugs that promote myelin development, many of which are conserved in mammalian glia. Recently, zebrafish have been employed as a model to understand the complex dynamics of myelinating glia during development and regeneration. In this chapter, we describe these key methodologies and recent insights into mechanisms that regulate myelination using the zebrafish model., (Copyright © 2017 Elsevier Inc. All rights reserved.)

The g-ratio, equal to the ratio of the inner-to-outer diameter of a myelinated axon, is associated with the speed of conduction, and thus reflects axonal function and integrity. It is now possible to estimate an "aggregate" g-ratio in vivo using MRI. The aim of this study was to assess the variation of the MRI-derived fiber g-ratio in the brain of healthy individuals, and to characterize its variation across the lifespan. Thirty-eight healthy participants, aged between 20 and 76, were recruited. Whole-brain g-ratio maps were computed and analyzed voxel-wise. Median tract g-ratio values were also extracted. No significant effect of gender was found, whereas age was found to be significantly associated with the g-ratio within the white matter. The tract-specific analysis showed this relationship to follow a nearly-linear increase, although the slope appears to slow down slightly after the 6th decade of life. The most likely interpretation is a subtle but consistent reduction in myelin throughout adulthood, with the density of axons beginning to decrease between the 4th and 5th decade., (Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.)

Oligodendrocytes myelinate neuronal axons during development and increase conduction velocity of neuronal impulses in the central nervous system. Neuronal axons extend from multiple brain regions and pass through the white matter; however, whether oligodendrocytes ensheath a particular set of axons or do so randomly within the mammalian brain remains unclear. We developed a novel method to visualize individual oligodendrocytes and axon derived from a particular brain region in mouse white matter using a combinational injection of attenuated rabies virus and adeno-associated virus. Using this method, we found that some populations of oligodendrocytes in the corpus callosum predominantly ensheathed axons derived from motor cortex or sensory cortex, while others ensheathed axons from both brain regions, suggesting heterogeneity in preference of myelination toward a particular subtype of neurons. Moreover, our newly established method is a versatile tool for analyzing precise morphology of each oligodendrocyte in animal models for demyelinating disorders and addressing the role of oligodendrocyte in higher brain functions. GLIA 2016. GLIA 2017;65:93-105., (© 2016 Wiley Periodicals, Inc.)

Charcot-Marie-Tooth 1A (CMT1A) is a demyelinating hereditary neuropathy for which pharmacological treatments are not yet available. An abnormally high intracellular Ca(2+) concentration was observed in Schwann cells (SC) from CMT1A rats, caused by the PMP22-mediated overexpression of the P2X7 purinoceptor. The purpose of this study was to investigate the tolerability and therapeutic potential of a pharmacological antagonist of the P2X7 receptor (A438079) in CMT1A. A438079 ameliorated in vitro myelination of organotypic DRG cultures from CMT1A rats. Furthermore, we performed an experimental therapeutic trial in PMP22 transgenic and in wild-type rats. A preliminary dose-escalation trial showed that 3mg/kg A438079 administered via intraperitoneal injection every 24h for four weeks was well tolerated by wild type and CMT1A rats. Affected rats treated with 3mg/kg A438079 revealed a significant improvement of the muscle strength, when compared to placebo controls. Importantly, histologic analysis revealed a significant increase of the total number of myelinated axons in tibial nerves. Moreover, a significant decrease of the hypermyelination of small caliber axons and a significant increase of the frequency and diameter of large caliber myelinated axons was highlighted. An improved distal motor latencies was recorded, whereas compound muscle action potentials (CMAP) remained unaltered. A438079 reduced the SC differentiation defect in CMT1A rats. These results show that pharmacological inhibition of the P2X7 receptor is well tolerated in CMT1A rats and represents a proof-of-principle that antagonizing this pathway may correct the molecular derangements and improve the clinical phenotype in the CMT1A neuropathy., (Copyright © 2016. Published by Elsevier Inc.)

Myelination is governed by neuron-glia communication, which in turn is modulated by neural activity. The exact mechanisms remain elusive. We developed a novel in vitro optogenetic stimulation platform that facilitates subcellular activity induction in hundreds of neurons simultaneously. The light isolation was achieved by creating a biocompatible, light-absorbent, black microfluidic device integrated with a programmable, high-power LED array. The system was applied to a compartmentalized culture of primary neurons whose distal axons were interacting with oligodendrocyte precursor cells. Neural activity was induced along whole neurons or was constrained to cell bodies with proximal axons or distal axons only. All three modes of stimulation promoted oligodendrocyte differentiation and the myelination of axons as evidenced by a decrease in the number of oligodendrocyte precursor cells followed by increases in the number of mature oligodendrocytes and myelin sheath fragments. These results demonstrated the potential of our novel optogenetic stimulation system for the global and focal induction of neural activity in vitro for studying axon myelination.

Myelination, the process by which oligodendrocytes form the myelin sheath around axons, is key to axonal signal transduction and related motor function in the central nervous system (CNS). Aging is characterized by degenerative changes in the myelin sheath, although the molecular underpinnings of normal and aberrant myelination remain incompletely understood. Here we report that axon myelination and related motor function are dependent on BubR1, a mitotic checkpoint protein that has been linked to progeroid phenotypes when expressed at low levels and healthy lifespan when overabundant. We found that oligodendrocyte progenitor cell proliferation and oligodendrocyte density is markedly reduced in mutant mice with low amounts of BubR1 ( BubR1 mice), causing axonal hypomyelination in both brain and spinal cord. Expression of essential myelin-related genes such as MBP and PLP1 was significantly reduced in these tissues. Consistent with defective myelination, H/H mice exhibited various motor deficits, including impaired motor strength, coordination, and balance, irregular gait patterns and reduced locomotor activity. Collectively, these data suggest that BubR1 is a key determinant of oligodendrocyte production and function and provide a molecular entry point to understand age-related degenerative changes in axon myelination.BubR1 H/H mice exhibited various motor deficits, including impaired motor strength, coordination, and balance, irregular gait patterns and reduced locomotor activity. Collectively, these data suggest that BubR1 is a key determinant of oligodendrocyte production and function and provide a molecular entry point to understand age-related degenerative changes in axon myelination., Competing Interests: The authors declare no competing financial interests.

Background: Regeneration of new myelin is impaired in persistent multiple sclerosis (MS) lesions, leaving neurons unable to function properly and subject to further degeneration. Current MS therapies attempt to ameliorate autoimmune-mediated demyelination, but none directly promote the regeneration of lost and damaged myelin of the central nervous system (CNS). Development of new drugs that stimulate remyelination has been hampered by the inability to evaluate axonal myelination in a rapid CNS culture system., Results: We established a high throughput cell-based assay to identify compounds that promote myelination. Culture methods were developed for initiating myelination in vitro using primary embryonic rat cortical cells. We developed an immunofluorescent phenotypic image analysis method to quantify the morphological alignment of myelin characteristic of the initiation of myelination. Using γ-secretase inhibitors as promoters of myelination, the optimal growth, time course and compound treatment conditions were established in a 96 well plate format. We have characterized the cortical myelination assay by evaluating the cellular composition of the cultures and expression of markers of differentiation over the time course of the assay. We have validated the assay scalability and consistency by screening the NIH clinical collection library of 727 compounds and identified ten compounds that promote myelination. Half maximal effective concentration (EC50) values for these compounds were determined to rank them according to potency., Conclusions: We have designed the first high capacity in vitro assay that assesses myelination of live axons. This assay will be ideal for screening large compound libraries to identify new drugs that stimulate myelination. Identification of agents capable of promoting the myelination of axons will likely lead to the development of new therapeutics for MS patients.

Background: Spinal cord injury (SCI) is a debilitating event with multiple mechanisms of degeneration leading to life-long paralysis. Biomaterial strategies, including bridges that span the injury and provide a pathway to reconnect severed regions of the spinal cord, can promote partial restoration of motor function following SCI. Axon growth through the bridge is essential to characterizing regeneration, as recovery can occur via other mechanisms such as plasticity. Quantitative analysis of axons by manual counting of histological sections can be slow, which can limit the number of bridge designs evaluated. In this study, we report a semi-automated process to resolve axon numbers in histological sections, which allows for efficient analysis of large data sets., New Method: Axon numbers were estimated in SCI cross-sections from animals implanted with poly(lactide co-glycolide) (PLG) bridges with multiple channels for guiding axons. Immunofluorescence images of histological sections were filtered using a Hessian-based approach prior to threshold detection to improve the signal-to-noise ratio and filter out background staining associated with PLG polymer., Results: Semi-automated counting successfully recapitulated average axon densities and myelination in a blinded PLG bridge implantation study., Comparison With Existing Methods: Axon counts obtained with the semi-automated technique correlated well with manual axon counts from blinded independent observers across sections with a wide range of total axons., Conclusions: This semi-automated detection of Hessian-filtered axons provides an accurate and significantly faster alternative to manual counting of axons for quantitative analysis of regeneration following SCI., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Niemann-Pick type C (NPC) disease is a genetic disorder associated with intracellular cholesterol accumulation in the brain and other organs, and neurodegeneration is generally believed to be the fatal cause of the disease. In view of the emerging role of matrix metalloproteinase-12 (MMP-12) in neuronal injury, we investigated its expression and potential roles in axonal degeneration in Npc1-/- mouse brain. Microarray and quantitative real-time reversed transcription PCR analysis indicated a marked increase in MMP-12 mRNA levels in cerebellum of 3 week-old Npc1-/- mice, as compared to wild-type littermates. Western blots showed that the ratio of mature MMP-12 over pro-MMP-12 was significantly increased in cerebellum of Npc1-/-, as compared to wild-type mice. Immunohistochemical studies confirmed that MMP-12 expression was increased, especially in the cell bodies of Purkinje neurons in Npc1-/- mice. Neuritic growth was significantly reduced by Npc1 siRNA knockdown in nerve growth factor-differentiated PC-12 cells, and this effect was completely reversed by treatment with an MMP-12 specific inhibitor. Furthermore, in vivo experiments showed that chronic treatment with the MMP-12 inhibitor ameliorated Npc1 deficiency-induced axonal pathology in the striatum. Our results indicate that abnormal neuronal expression of MMP-12 may contribute to axonal degeneration in NPC disease, thus providing a potential novel target for treatment., (Copyright © 2015 Elsevier Inc. All rights reserved.)

In the central nervous system (CNS), myelin is formed by oligodendrocytes that are derived from precursor cells, known as oligodendrocyte precursor cells (OPCs). Successive stages of OPC interactions with the axons can be visualized in vitro and ex vivo using mixed neural cell cultures and pieces of intact spinal cord, respectively. OPCs and their differentiation can be imaged using cell-type-specific markers or green fluorescent protein (GFP) tags. This protocol describes methodology for generating these two systems for time-lapse imaging of dynamic cell interactions using fluorescent and 2-photon microscopy., (Copyright © 2015 John Wiley & Sons, Inc.)

In this article we discuss the molecular signaling mechanisms that coordinate interactions between Schwann cells and the neurons of the peripheral nervous system. Such interactions take place perpetually during development and in adulthood, and are critical for the homeostasis of the peripheral nervous system (PNS). Neurons provide essential signals to control Schwann cell functions, whereas Schwann cells promote neuronal survival and allow efficient transduction of action potentials. Deregulation of neuron-Schwann cell interactions often results in developmental abnormalities and diseases. Recent investigations have shown that during development, neuronally provided signals, such as Neuregulin, Jagged, and Wnt interact to fine-tune the Schwann cell lineage progression. In adult, the signal exchange between neurons and Schwann cells ensures proper nerve function and regeneration. Identification of the mechanisms of neuron-Schwann cell interactions is therefore essential for our understanding of the development, function and pathology of the peripheral nervous system as a whole., (© 2015 WILEY Periodicals, Inc.)

The present study investigated the effectiveness of mesenchymal stem cells (MSCs) associated with a fibrin scaffold (FS) for the peripheral regenerative process after nerve tubulization. Adult female Lewis rats received a unilateral sciatic nerve transection followed by repair with a polycaprolactone (PCL)-based tubular prosthesis. Sixty days after injury, the regenerated nerves were studied by immunohistochemistry. Anti-p75NTR immunostaining was used to investigate the reactivity of the MSCs. Basal labeling, which was upregulated during the regenerative process, was detected in uninjured nerves and was significantly greater in the MSC-treated group. The presence of GFP-positive MSCs was detected in the nerves, indicating the long term survival of such cells. Moreover, there was co-localization between MSCs and BNDF immunoreactivity, showing a possible mechanism by which MSCs improve the reactivity of SCs. Myelinated axon counting and morphometric analyses showed that MSC engrafting led to a higher degree of fiber compaction combined with a trend of increased myelin sheath thickness, when compared with other groups. The functional result of MSC engrafting was that the animals showed higher motor function recovery at the seventh and eighth week after lesion. The findings herein show that MSC+FS therapy improves the nerve regeneration process by positively modulating the reactivity of SCs., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Schwann cells (SCs) have been considered to be one of the most promising cell types for transplantation to treat spinal cord injury (SCI) due to their unique growth-promoting properties. Despite the extensive use as donor cells for transplantation in SCI models, the fate of SCs is controversial due in part to the lack of a reliable marker for tracing the grafted SCs. To precisely assess the fate and temporal profile of transplanted SCs, we isolated purified SCs from sciatic nerves of adult transgenic rats overexpressing GFP (SCs-GFP). SCs-GFP were directly injected into the epicenter of a moderate contusive SCI at the mid-thoracic level at 1week post-injury. The number of SCs-GFP or SCs-GFP labeled with Bromodeoxyuridine (BrdU) was quantified at 5min, 1day, and 1, 2, 4, 12 and 24weeks after cell injection. Basso, Beattie, and Bresnahan (BBB) locomotor rating scale, footfall error, thermal withdrawal latency, and footprint analysis were performed before and after the SCs-GFP transplantation. After transplantation, SCs-GFP quickly filled the lesion cavity. A remarkable survival of grafted SCs-GFP up to 24weeks post-grafting was observed with clearly identified SC individuals. SCs-GFP proliferated after injection, peaked at 2weeks (26% of total SCs-GFP), decreased thereafter, and ceased at 12weeks post-grafting. Although grafted SCs-GFP were mainly confined within the border of surrounding host tissue, they migrated along the central canal for up to 5.0mm at 4weeks post-grafting. Within the lesion site, grafted SCs-GFP myelinated regenerated axons and expressed protein zero (P0) and myelin basic protein (MBP). Within the SCs-GFP grafts, new blood vessels were formed. Except for a significant decrease of angle of rotation in the footprint analysis, we did not observe significant behavioral improvements in BBB locomotor rating scale, thermal withdrawal latency, or footfall errors, compared to the control animals that received no SCs-GFP. We conclude that SCs-GFP can survive remarkably well, proliferate, migrate along the central canal, and myelinate regenerated axons when being grafted into a clinically-relevant contusive SCI in adult rats. Combinatorial strategies, however, are essential to achieve a more meaningful functional regeneration of which SCs may play a significant role., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Oligodendrocytes and the myelin they produce are a remarkable vertebrate specialization that enables rapid and efficient nerve conduction within the central nervous system. The generation of myelin during development involves a finely-tuned pathway of oligodendrocyte precursor specification, proliferation and migration followed by differentiation and the subsequent myelination of appropriate axons. In this review we summarize the molecular mechanisms known to regulate each of these processes, including the extracellular ligands that promote or inhibit development of the oligodendrocyte lineage, the intracellular pathways they signal through and the key transcription factors that mediate their effects. Many of these regulatory mechanisms have recurring roles in regulating several transitions during oligodendrocyte development, highlighting their importance. It is also highly likely that many of these developmental mechanisms will also be involved in myelin repair in human neurological disease., (Copyright © 2013 IBRO. Published by Elsevier Ltd. All rights reserved.)

CNS white matter is subject to a novel form of neural plasticity which has been termed "myelin plasticity". It is well established that oligodendrocyte generation and the addition of new myelin internodes continue throughout normal adulthood. These new myelin internodes maybe required for the de novo myelination of previously unmyelinated axons, myelin sheath replacement, or even myelin remodeling. Each process could alter axonal conduction velocity, but to what end? We review the changes that occur within the white matter over the lifetime, the known regulators and mediators of white matter plasticity in the mature CNS, and the physiological role this plasticity may play in CNS function., (Copyright © 2013 IBRO. Published by Elsevier Ltd. All rights reserved.)

About half of the human brain is white matter, characterized by axons covered in myelin, which facilitates the high speed of nerve signals from one brain area to another. At the time of myelination, the oligodendrocytes that synthesize myelin require a large amount of energy for this task. Conditions that deprive the tissue of energy can kill the oligodendrocytes. During brain development, the oligodendrocytes may use lactate as an alternative source of energy and material for myelin formation. Mature oligodendrocytes, however, can release lactate through the myelin sheath as nutrient for axons. In addition, lactate carries signals as a volume transmitter. Myelin thus seems to serve as a provider of substrates and signals for axons, and not as a mere insulator. We review the fluxes of lactate in white matter and their significance in brain function., (Copyright © 2013 IBRO. Published by Elsevier Ltd. All rights reserved.)

Neural precursor cells (NPCs) offer a promising approach for treating demyelinating diseases. However, the cellular dynamics that underlie transplanted NPC-mediated remyelination have not been described. Using two-photon imaging of a newly developed ventral spinal cord preparation and a viral model of demyelination, we describe the motility and intercellular interactions of transplanted mouse NPCs expressing green fluorescent protein (GFP) with damaged axons expressing yellow fluorescent protein (YFP). Our findings reveal focal axonal degeneration that occurs in the ventral side of the spinal cord within 1 wk following intracranial instillation with the neurotropic JHM strain of mouse hepatitis virus (JHMV). Axonal damage precedes extensive demyelination and is characterized by swelling along the length of the axon, loss of YFP signal, and transected appearance. NPCs engrafted into spinal cords of JHMV-infected mice exhibited diminished migration velocities and increased proliferation compared with transplanted cells in noninfected mice. NPCs preferentially accumulated within areas of axonal damage, initiated direct contact with axons, and subsequently expressed the myelin proteolipid protein gene, initiating remyelination. These findings indicate that NPCs transplanted into an inflammatory demyelinating microenvironment participate directly in therapeutic outcome through the wrapping of myelin around damaged neurons.

Amyloid precursor protein (APP), commonly associated with Alzheimer disease, is upregulated and distributes evenly along the injured axons, and therefore, also known as a marker of demyelinating axonal injury and axonal degeneration. However, the physiological distribution and function of APP along myelinated axons was unknown. We report that APP aggregates at nodes of Ranvier (NOR) in the myelinated central nervous system (CNS) axons but not in the peripheral nervous system (PNS). At CNS NORs, APP expression co-localizes with tenascin-R and is flanked by juxtaparanodal potassium channel expression demonstrating that APP localized to NOR. In APP-knockout (KO) mice, nodal length is significantly increased, while sodium channels are still clustered at NORs. Moreover, APP KO and APP-overexpressing transgenic (APP TG) mice exhibited a decreased and an increased thickness of myelin in spinal cords, respectively, although the changes are limited in comparison to their littermate WT mice. The thickness of myelin in APP KO sciatic nerve also increased in comparison to that in WT mice. Our observations indicate that APP acts as a novel component at CNS NORs, modulating nodal formation and has minor effects in promoting myelination.

Phosphoinositide 3-kinase γ (PI3Kγ) is a shared downstream component of chemokine-mediated signaling pathways and regulates migration, proliferation and activation of inflammatory cells. PI3Kγ has been shown to play a crucial role in regulating inflammatory responses during the progression of several diseases. We investigated the potential function of PI3Kγ in mediating inflammatory reactions and the development of experimental autoimmune encephalomyelitis (EAE), a model of multiple sclerosis (MS). We found that systemic treatment with selective PI3Kγ inhibitor AS-604850 significantly reduced the number of infiltrated leukocytes in the CNS and ameliorated the clinical symptoms of EAE mice. Treatment with this PI3Kγ inhibitor enhanced myelination and axon number in the spinal cord of EAE mice. Consistently, we demonstrated that PI3Kγ deletion in knockout mice mitigates the clinical sign of EAE compared to PI3Kγ+/+ controls. PI3Kγ deletion increased the number of axons in the lumbar spinal cord, including descending 5-HT-positive serotonergic fiber tracts. Our results indicate that PI3Kγ contributes to development of autoimmune CNS inflammation and that PI3Kγ blockade may provide a great potential for treating patients with MS., (Copyright © 2013 IBRO. Published by Elsevier Ltd. All rights reserved.)

Purpose: In most mammalian species, retinal ganglion cell (RGC) axons are myelinated in the optic nerve, but remain nonmyelinated in the retinal nerve fiber layer and the most proximal (i.e., retina-near) region of the nerve. Here we analyzed whether RGCs are involved in the control of this characteristic distribution of oligodendrocytes and myelin in the primary visual pathway of mice., Methods: Neurospheres were enriched in oligodendrocyte progenitor cells (OPCs) by a short-term exposure to platelet-derived growth factor (PDGF) and grafted into the retina of young postnatal mice close to the optic disc. Immunohistochemistry was performed to study the integration and differentiation of the grafted cells, and the formation of donor-derived myelin in the normally nonmyelinated retinal nerve fiber layer and intrabulbar and most proximal retrobulbar region of the optic nerve., Results: Intraretinal transplantations of small-sized PDGF-treated neurospheres into young postnatal mice resulted in extensive integration of the grafted cells into host retinas. A significant fraction of the donor cells differentiated into oligodendrocytes that myelinated the nerve fiber layer. Importantly, RGC axon segments within the normally nonmyelinated intrabulbar and most proximal retrobulbar region of the nerve also became myelinated in a fraction of animals., Conclusions: This is the first report demonstrating that the normally nonmyelinated intrabulbar and retrobulbar segments of RGC axons are competent to become myelinated. Results support the view that the differential distribution of myelin and oligodendrocytes in the primary visual pathway is controlled by nonneuronal factors rather than by the RGCs themselves.

Schwann cell (SC) migration is an important step preceding myelination and remyelination in the peripheral nervous system, and can be promoted by peptide factors like neuregulins. Here we present evidence that a lipid factor, lysophosphatidic acid (LPA), influences both SC migration and peripheral myelination through its cognate G protein-coupled receptor (GPCR) known as LPA1 . Ultrastructural analyses of peripheral nerves in mouse null-mutants for LPA1 showed delayed SC-to-axon segregation, polyaxonal myelination by single SCs, and thinner myelin sheaths. In primary cultures, LPA promoted SC migration through LPA1 , while analysis of conditioned media from purified dorsal root ganglia neurons using HPLC/MS supported the production of LPA by these neurons. The heterotrimeric G-alpha protein, Gαi , and the small GTPase, Rac1, were identified as important downstream signaling components of LPA1 . These results identify receptor mediated LPA signaling between neurons and SCs that promote SC migration and contribute to the normal development of peripheral nerves through effects on SC-axon segregation and myelination., (Copyright © 2013 Wiley Periodicals, Inc.)

During late Schwann cell development, immature Schwann cells segregate large axons from bundles, a process called "axonal radial sorting." Here we demonstrate that canonical Wnt signals play a critical role in radial sorting and assign a role to Wnt and Rspondin ligands in this process. Mice carrying β-catenin loss-of-function mutations show a delay in axonal sorting; conversely, gain-of-function mutations result in accelerated sorting. Sorting deficits are accompanied by abnormal process extension, differentiation, and aberrant cell cycle exit of the Schwann cells. Using primary cultured Schwann cells, we analyze the upstream effectors, Wnt and Rspondin ligands that initiate signaling, and downstream genetic programs that mediate the Wnt response. Our analysis contributes to a better understanding of the mechanisms of Schwann cell development and fate decisions.

Regeneration in the injured spinal cord is limited by physical and chemical barriers. Acute implantation of a multichannel poly(lactide-co-glycolide) (PLG) bridge mechanically stabilizes the injury, modulates inflammation, and provides a permissive environment for rapid cellularization and robust axonal regrowth through this otherwise inhibitory milieu. However, without additional intervention, regenerated axons remain largely unmyelinated (<10%), limiting functional repair. While transplanted human neural stem cells (hNSC) myelinate axons after spinal cord injury (SCI), hNSC fate is highly influenced by the SCI inflammatory microenvironment, also limiting functional repair. Accordingly, we investigated the combination of PLG scaffold bridges with hNSC to improve histological and functional outcome after SCI. In vitro, hNSC culture on a PLG scaffold increased oligodendroglial lineage selection after inflammatory challenge. In vivo, acute PLG bridge implantation followed by chronic hNSC transplantation demonstrated a robust capacity of donor human cells to migrate into PLG bridge channels along regenerating axons and integrate into the host spinal cord as myelinating oligodendrocytes and synaptically integrated neurons. Axons that regenerated through the PLG bridge formed synaptic circuits that connected the ipsilateral forelimb muscle to contralateral motor cortex. hNSC transplantation significantly enhanced the total number of regenerating and myelinated axons identified within the PLG bridge. Finally, the combination of acute bridge implantation and hNSC transplantation exhibited robust improvement in locomotor recovery. These data identify a successful strategy to enhance neurorepair through a temporally layered approach using acute bridge implantation and chronic cell transplantation to spare tissue, promote regeneration, and maximize the function of new axonal connections. [ABSTRACT FROM AUTHOR]