Hisami Koito

The oligodendrocyte-specific G-protein coupled receptor GPR17 is a cell-intrinsic timer of myelination

The basic helix-loop-helix transcription factor Olig1 promotes oligodendrocyte maturation and is required for myelin repair. We characterized an Olig1-regulated G protein–coupled receptor, GPR17, whose function is to oppose the action of Olig1. Gpr17 was restricted to oligodendrocyte lineage cells, but was downregulated during the peak period of myelination and in adulthood. Transgenic mice with sustained Gpr17 expression in oligodendrocytes exhibited stereotypic features of myelinating disorders in the CNS. Gpr17 overexpression inhibited oligodendrocyte differentiation and maturation both in vivo and in vitro. Conversely, Gpr17 knockout mice showed early onset of oligodendrocyte myelination. The opposing action of Gpr17 on oligodendrocyte maturation reflects, at least partially, upregulation and nuclear translocation of the potent oligodendrocyte differentiation inhibitors ID2/4. Collectively, these findings suggest that GPR17 orchestrates the transition between immature and myelinating oligodendrocytes via an ID protein–mediated negative regulation and may serve as a potential therapeutic target for CNS myelin repair.

Tumor necrosis factor alpha mediates lipopolysaccharide-induced microglial toxicity to developing oligodendrocytes when astrocytes are present.

Reactive microglia and astrocytes are present in lesions of white matter disorders, such as periventricular leukomalacia and multiple sclerosis. However, it is not clear whether they are actively involved in the pathogenesis of these disorders. Previous studies demonstrated that microglia, but not astrocytes, are required for lipopolysaccharide (LPS)-induced selective killing of developing oligodendrocytes (preOLs) and that the toxicity is mediated by microglia-derived peroxynitrite. Here we report that, when astrocytes are present, the LPS-induced, microglia-dependent toxicity to preOLs is no longer mediated by peroxynitrite but instead by a mechanism dependent on tumor necrosis factor-α (TNFα) signaling. Blocking peroxynitrite formation with nitric oxide synthase (NOS) inhibitors or a decomposition catalyst did not prevent LPS-induced loss of preOLs in mixed glial cultures. PreOLs were highly vulnerable to peroxynitrite; however, the presence of astrocytes prevented the toxicity. Whereas LPS failed to kill preOLs in cocultures of microglia and preOLs deficient in inducible NOS (iNOS) or gp91phox, the catalytic subunit of the superoxide-generating NADPH oxidase, LPS caused a similar degree of preOL death in mixed glial cultures of wild-type, iNOS−/−, and gp91phox−/− mice. TNFα neutralizing antibody inhibited LPS toxicity, and addition of TNFα induced selective preOL injury in mixed glial cultures. Furthermore, disrupting the genes encoding TNFα or its receptors TNFR1/2 completely abolished the deleterious effect of LPS. Our results reveal that TNFα signaling, rather than peroxynitrite, is essential in LPS-triggered preOL death in an environment containing all major glial cell types and underscore the importance of intercellular communication in determining the mechanism underlying inflammatory preOL death.

Microfluidic compartmentalized co-culture platform for CNS axon myelination research

This paper presents a circular microfluidic compartmentalized co-culture platform that can be used for central nervous system (CNS) axon myelination research. The microfluidic platform is composed of a soma compartment and an axon/glia compartment connected through arrays of axon-guiding microchannels. Myelin-producing glia, oligodendrocytes (OLs), placed in the axon/glia compartment, interact with only axons but not with neuronal somata confined to the soma compartment, reminiscent to in vivo situation where many axon fibres are myelinated by OLs at distance away from neuronal cell bodies. Primary forebrain neurons from embryonic day 16–18 rats were cultured inside the soma compartment for two weeks to allow them to mature and form extensive axon networks. OL progenitors, isolated from postnatal day 1-2 rat brains, were then added to the axon/glia compartment and co-cultured with neurons for an additional two weeks. The microdevice showed fluidic isolation between the two compartments and successfully isolated neuronal cell bodies and dendrites from axons growing through the arrays of axon-guiding microchannels into the axon/glia compartment. The circular co-culture device developed here showed excellent cell loading characteristics where significant numbers of cells were positioned near the axon-guiding microchannels. This significantly increased the probability of axons crossing these microchannels as demonstrated by the more than 51 % of the area of the axon/glia compartment covered with axons two weeks after cell seeding. OL progenitors co-cultured with axons inside the axon/glia compartment successfully differentiated into mature OLs. These results indicate that this device can be used as an excellent in vitro co-culture platform for studying localized axon-glia interaction and signalling.

Astrocytes promote TNF‐mediated toxicity to oligodendrocyte precursors

J. Neurochem. (2011) 116, 53–66.

Activation of oligodendroglial Stat3 is required for efficient remyelination

Multiple sclerosis is the most prevalent demyelinating disease of the central nervous system (CNS) and is histologically characterized by perivascular demyelination as well as neurodegeneration. While the degree of axonal damage is correlated with clinical disability, it is believed that remyelination can protect axons from degeneration and slow disease progression. Therefore, understanding the intricacies associated with myelination and remyelination may lead to therapeutics that can enhance the remyelination process and slow axon degeneration and loss of function. Ciliary neurotrophic factor (CNTF) family cytokines such as leukemia inhibitory factor (LIF) and interleukin 11 (IL-11) are known to promote oligodendrocyte maturation and remyelination in experimental models of demyelination. Because CNTF family member binding to the gp130 receptor results in activation of the JAK2/Stat3 pathway we investigated the necessity of oligodendroglial Stat3 in transducing the signal required for myelination and remyelination. We found that Stat3 activation in the CNS coincides with myelination during development. Stimulation of oligodendrocyte precursor cells (OPCs) with CNTF or LIF promoted OPC survival and final differentiation, which was completely abolished by pharmacologic blockade of Stat3 activation with JAK2 inhibitor. Similarly, genetic ablation of Stat3 in oligodendrocyte lineage cells prevented CNTF-induced OPC differentiation in culture. In vivo, while oligodendroglial Stat3 signaling appears to be dispensable for developmental CNS myelination, it is required for oligodendrocyte regeneration and efficient remyelination after toxin-induced focal demyelination in the adult brain. Our data suggest a critical function for oligodendroglial Stat3 signaling in myelin repair.

A Multi-compartment CNS Neuron-glia Co-culture Microfluidic Platform

We present a novel multi-compartment neuron co-culture microsystem platform for in vitro CNS axon-glia interaction research, capable of conducting up to six independent experiments in parallel for higher-throughput. We developed a new fabrication method to create microfluidic devices having both micro and macro scale structures within the same device through a single soft-lithography process, enabling mass fabrication with good repeatability. The multi-compartment microfluidic co-culture platform is composed of one soma compartment for neurons and six axon/glia compartments for oligodendrocytes (OLs). The soma compartment and axon/glia compartments are connected by arrays of axon-guiding microchannels that function as physical barriers to confine neuronal soma in the soma compartment, while allowing axons to grow into axon/glia compartments. OLs loaded into axon/glia compartments can interact only with axons but not with neuronal soma or dendrites, enabling localized axon-glia interaction studies. The microchannels also enabled fluidic isolation between compartments, allowing six independent experiments to be conducted on a single device for higher throughput. Soft-lithography using poly(dimethylsiloxane) (PDMS) is a commonly used technique in biomedical microdevices. Reservoirs on these devices are commonly defined by manual punching. Although simple, poor alignment and time consuming nature of the process makes this process not suitable when large numbers of reservoirs have to be repeatedly created. The newly developed method did not require manual punching of reservoirs, overcoming such limitations. First, seven reservoirs (depth: 3.5 mm) were made on a poly(methyl methacrylate) (PMMA) block using a micro-milling machine. Then, arrays of ridge microstructures, fabricated on a glass substrate, were hot-embossed against the PMMA block to define microchannels that connect the soma and axon/glia compartments. This process resulted in macro-scale reservoirs (3.5 mm) and micro-scale channels (2.5 microm) to coincide within a single PMMA master. A PDMS replica that served as a mold master was obtained using soft-lithography and the final PDMS device was replicated from this master. Primary neurons from E16-18 rats were loaded to the soma compartment and cultured for two weeks. After one week of cell culture, axons crossed microchannels and formed axonal only network layer inside axon/glia compartments. Axons grew uniformly throughout six axon/glia compartments and OLs from P1-2 rats were added to axon/glia compartments at 14 days in vitro for co-culture.

Preparation of Rat Brain Aggregate Cultures for Neuron and Glia Development Studies

An in vitro system that recapitulates the development and differentiation of progenitors into mature neurons and glia in the central nervous system (CNS) would provide a powerful platform for neuroscientists to investigate axo-glial interactions, properties and differentiation of multipotent progenitors, and progression of oligodendroglial lineage cells at the cellular and molecular level. We describe here a CNS aggregate culture system from embryonic rat forebrains, which can be maintained in a serum-free medium up to 3-4 weeks and is used in our laboratory as a model to study neuron-glia interaction and CNS myelination. This video clip will demonstrate how to isolate and grow these CNS aggregate cultures from E16 rat brain. Furthermore, from the same brain dissection, highly enriched regular dissociated neuronal cultures can be readily obtained and used for various studies on CNS neurons or used for co-cultures with other cells.