Current methods for studying central nervous system myelination necessitate permissive axonal substrates conducive for myelin wrapping by oligodendrocytes. We have developed a neuron-free culture system in which electron-spun nanofibers of varying sizes substitute for axons as a substrate for oligodendrocyte myelination, thereby allowing manipulation of the biophysical elements of axonal-oligodendroglial interactions. To investigate axonal regulation of myelination, this system effectively uncouples the role of molecular (inductive) cues from that of biophysical properties of the axon. We use this method to uncover the causation and sufficiency of fiber diameter in the initiation of concentric wrapping by rat oligodendrocytes. We also show that oligodendrocyte precursor cells display sensitivity to the biophysical properties of fiber diameter and initiate membrane ensheathment prior to differentiation. The use of nanofiber scaffolds will enable screening for potential therapeutic agents that promote oligodendrocyte differentiation and myelination as well as provide valuable insight into the processes involved in remyelination.
Current methods for studying oligodendrocyte myelination using primary neurons are limited by the time, cost and reproducibility of myelination in vitro. Nanofibers with diameters of >0.4 μm fabricated from electrospinning of liquid polystyrene are suitable scaffolds for concentric membrane wrapping by oligodendrocytes. With the advent of aligned electrospinning technology, nanofibers can be rapidly fabricated, standardized, and configured into various densities and patterns as desired. Notably, the minimally permissive culture environment of fibers provides investigators with an opportunity to explore the autonomous oligodendrocyte cellular processes underlying differentiation and myelination. The simplicity of the system is conducive to monitoring oligodendrocyte proliferation, migration, differentiation and membrane wrapping in the absence of neuronal signals. Here we describe protocols for the fabrication and preparation of nanofibers aligned on glass coverslips for the study of membrane wrapping by rodent oligodendrocytes. The entire protocol can be completed within 2 weeks.
Multiple sclerosis (MS) is the most common multifocal inflammatory demyelinating disease of the central nervous system (CNS). Due to the progressive neurodegenerative nature of MS, developing treatments that exhibit direct neuroprotective effects are needed. Tecfidera™ (BG-12) is an oral formulation of the fumaric acid esters (FAE), containing the active metabolite dimethyl fumarate (DMF). Although BG-12 showed remarkable efficacy in lowering relapse rates in clinical trials, its mechanism of action in MS is not yet well understood. In this study, we reported the potential neuroprotective effects of dimethyl fumarate (DMF) on mouse and rat neural stem/progenitor cells (NPCs) and neurons. We found that DMF increased the frequency of the multipotent neurospheres and the survival of NPCs following oxidative stress with hydrogen peroxide (H2O2) treatment. In addition, utilizing the reactive oxygen species (ROS) assay, we showed that DMF reduced ROS production induced by H2O2. DMF also decreased oxidative stress-induced apoptosis. Using motor neuron survival assay, DMF significantly promoted survival of motor neurons under oxidative stress. We further analyzed the expression of oxidative stress-induced genes in the NPC cultures and showed that DMF increased the expression of transcription factor nuclear factor-erythroid 2-related factor 2 (Nrf2) at both levels of RNA and protein. Furthermore, we demonstrated the involvement of Nrf2-ERK1/2 MAPK pathway in DMF-mediated neuroprotection. Finally, we utilized SuperArray gene screen technology to identify additional anti-oxidative stress genes (Gstp1, Sod2, Nqo1, Srxn1, Fth1). Our data suggests that analysis of anti-oxidative stress mechanisms may yield further insights into new targets for treatment of multiple sclerosis (MS).
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