Spatial and Temporal Profiles of Growth Factor Expression during CNS Demyelination Reveal the Dynamics of Repair Priming

Demyelination is the cause of disability in various neurological disorders. It is therefore crucial to understand the molecular regulation of oligodendrocytes, the myelin forming cells in the CNS. Growth factors are known to be essential for the development and maintenance of oligodendrocytes and are involved in the regulation of glial responses in various pathological conditions. We employed the well established murine cuprizone model of toxic demyelination to analyze the expression of 13 growth factors in the CNS during de- and remyelination. The temporal mRNA expression profile during demyelination and the subsequent remyelination were analyzed separately in the corpus callosum and cerebral cortex using laser microdissection and real-time PCR techniques. During demyelination a similar pattern of growth factor mRNA expression was observed in both areas with a strong up-regulation of NRG1 and GDNF and a slight increase of CNTF in the first week of cuprizone treatment. HGF, FGF-2, LIF, IGF-I, and TGF-ß1 were up-regulated mainly during peak demyelination. In contrast, during remyelination there were regional differences in growth factor mRNA expression levels. GDNF, CNTF, HGF, FGF-2, and BDNF were elevated in the corpus callosum but not in the cortex, suggesting tissue differences in the molecular regulation of remyelination in the white and grey matter. To clarify the cellular source we isolated microglia from the cuprizone lesions. GDNF, IGF-1, and FGF mRNA were detected in the microglial fraction with a temporal pattern corresponding to that from whole tissue PCR. In addition, immunohistochemical analysis revealed IGF-1 protein expression also in the reactive astrocytes. CNTF was located in astrocytes. This study identified seven different temporal expression patterns for growth factors in white and grey matter and demonstrated the importance of early tissue priming and exact orchestration of different steps during callosal and cortical de- and remyelination

Microglia-mediated demyelination protects against CD8+ T cell-driven axon degeneration in mice carrying PLP defects

Axon degeneration and functional decline in myelin diseases are often attributed to loss of myelin but their relation is not fully understood. Perturbed myelinating glia can instigate chronic neuroinflammation and contribute to demyelination and axonal damage. Here we study mice with distinct defects in the proteolipid protein 1 gene that develop axonal damage which is driven by cytotoxic T cells targeting myelinating oligodendrocytes. We show that persistent ensheathment with perturbed myelin poses a risk for axon degeneration, neuron loss, and behavioral decline. We demonstrate that CD8(+) T cell-driven axonal damage is less likely to progress towards degeneration when axons are efficiently demyelinated by activated microglia. Mechanistically, we show that cytotoxic T cell effector molecules induce cytoskeletal alterations within myelinating glia and aberrant actomyosin constriction of axons at paranodal domains. Our study identifies detrimental axon-glia-immune interactions which promote neurodegeneration and possible therapeutic targets for disorders associated with myelin defects and neuroinflammation. Demyelination is often suggested to cause axonal degeneration. Here, the authors study mice carrying distinct PLP defects and reveal how persistent ensheathment with perturbed myelin poses a risk for CD8+T cell-driven axon loss and behavioral decline

Polysialic acid promotes remyelination in cerebellar slice cultures by Siglec-E-dependent modulation of microglia polarization

Multiple sclerosis is an inflammatory demyelinating disease of the central nervous system. Spontaneous restoration of myelin after demyelination occurs, but its efficiency declines during disease progression. Efficient myelin repair requires fine-tuning inflammatory responses by brain-resident microglia and infiltrating macrophages. Accordingly, promising therapeutic strategies aim at controlling inflammation to promote remyelination. Polysialic acid (polySia) is a polymeric glycan with variable chain lengths, presented as a posttranslational modification on select protein carriers. PolySia emerges as a negative regulator of inflammatory microglia and macrophage activation and has been detected on oligodendrocyte precursors and reactive astrocytes in multiple sclerosis lesions. As shown recently, polySia-modified proteins can also be released by activated microglia, and the intrinsically released protein-bound and exogenously applied free polySia were equally able to attenuate proinflammatory microglia activation via the inhibitory immune receptor Siglec-E. In this study, we explore polySia as a candidate substance for promoting myelin regeneration by immunomodulation. Lysophosphatidylcholine-induced demyelination of organotypic cerebellar slice cultures was used as an experimental model to analyze the impact of polySia with different degrees of polymerization (DP) on remyelination and inflammation. In lysophosphatidylcholine-treated cerebellar slice cultures, polySia-positive cells were abundant during demyelination but largely reduced during remyelination. Based on the determination of DP24 as the minimal polySia chain length required for the inhibition of inflammatory BV2 microglia activation, pools with short and long polySia chains (DP8–14 and DP24–30) were generated and applied to slice cultures during remyelination. Unlike DP8–14, treatment with DP24–30 significantly improved remyelination, increased arginase-1-positive microglia ratios, and reduced the production of nitric oxide in wildtype, but not in Siglec-E-deficient slice cultures. In vitro differentiation of oligodendrocytes was not affected by DP24–30. Collectively, these results suggest a beneficial effect of exogenously applied polySia DP24–30 on remyelination by Siglec-E-dependent microglia regulation

第925回千葉医学会例会・第20回千葉大学医学部放射線医学教室同門会例会

Teriflunomide does not affect cell death in cultured primary microglia. Isolated microglia were treated with LPS (100 ng/ml) and different concentrations of teriflunomide (0.25–5 μM) for 12, 48, or 72 h followed by staining with propidium iodide (PI). (A, B, C) The percentage of gated PI+ CD11b/c+ cells is presented as mean + SD (n = 4). (TIF 718 kb

Limited role of regulatory T cells during acute Theiler virus-induced encephalitis in resistant C57BL/6 mice

Background: Theiler's murine encephalomyelitis virus (TMEV) infection represents a commonly used infectious animal model to study various aspects of the pathogenesis of multiple sclerosis (MS). In susceptible SJL mice, dominant activity of Foxp3+ CD4+ regulatory T cells (Tregs) in the CNS partly contributes to viral persistence and progressive demyelination. On the other hand, resistant C57BL/6 mice rapidly clear the virus by mounting a strong antiviral immune response. However, very little is known about the role of Tregs in regulating antiviral responses during acute encephalitis in resistant mouse strains. Methods: In this study, we used DEREG mice that express the diphtheria toxin (DT) receptor under control of the foxp3 locus to selectively deplete Foxp3+ Tregs by injection of DT prior to infection and studied the effect of Treg depletion on the course of acute Theiler's murine encephalomyelitis (TME). Results: As expected, DEREG mice that are on a C57BL/6 background were resistant to TMEV infection and cleared the virus within days of infection, regardless of the presence or absence of Tregs. Nevertheless, in the absence of Tregs we observed priming of stronger effector T cell responses in the periphery, which subsequently resulted in a transient increase in the frequency of IFNγ-producing T cells in the brain at an early stage of infection. Histological and flow cytometric analysis revealed that this transiently increased frequency of brain-infiltrating IFNγ-producing T cells in Treg-depleted mice neither led to an augmented antiviral response nor enhanced inflammation-mediated tissue damage. Intriguingly, Treg depletion did not change the expression of IL-10 in the infected brain, which might play a role for dampening the inflammatory damage caused by the increased number of effector T cells. Conclusion: We therefore propose that unlike susceptible mice strains, interfering with the Treg compartment of resistant mice only has negligible effects on virus-induced pathologies in the CNS. Furthermore, in the absence of Tregs, local anti-inflammatory mechanisms might limit the extent of damage caused by strong anti-viral response in the CNS

De- and remyelination in the CNS white and grey matter induced by cuprizone: The old, the new, and the unexpected

The copper chelator cuprizone (bis-cyclohexanone oxaldihydrazone) was established as a neurotoxin in rodents in 1966 by Carlton. During the following years the usefulness of cuprizone feeding in mice to induce oligodendrocyte death with secondary demyelination of the superior cerebellar peduncles was described by Blakemore. In 1998 the cuprizone model experienced a renaissance as the group of Matsushima described the effects of cuprizone on the white matter of the cerebrum and focussed on demyelination in the corpus callosum, where the extent of demyelination could be scored more easily and consistently. Since then the toxic cuprizone model has been widely used to study experimental de- and remyelination in the corpus callosum. Recently, we and others have extended these studies and have shown several new aspects characteristic for this model. Many lessons can be learned from these recent findings that have implications for the basic understanding of remyelination and the design of remyelinating and neuroprotective strategies in demyelinating diseases of the CNS. Although the model is often mentioned in the context of multiple sclerosis, it must always be kept in mind that this model has a fundamentally different induction of demyelination. We highlight the important findings delineated from this model and critically discuss both the advantages and the shortcomings of cuprizone induced demyelination

Regenerative Effects of CDP-Choline: A Dose-Dependent Study in the Toxic Cuprizone Model of De- and Remyelination

Inflammatory attacks and demyelination in the central nervous system (CNS) are the key factors responsible for the damage of neurons in multiple sclerosis (MS). Remyelination is the natural regenerating process after demyelination that also provides neuroprotection but is often incomplete or fails in MS. Currently available therapeutics are affecting the immune system, but there is no substance that might enhance remyelination. Cytidine-S-diphosphate choline (CDP-choline), a precursor of the biomembrane component phospholipid phosphatidylcholine was shown to improve remyelination in two animal models of demyelination. However, the doses used in previous animal studies were high (500 mg/kg), and it is not clear if lower doses, which could be applied in human trials, might exert the same beneficial effect on remyelination. The aim of this study was to confirm previous results and to determine the potential regenerative effects of lower doses of CDP-choline (100 and 50 mg/kg). The effects of CDP-choline were investigated in the toxic cuprizone-induced mouse model of de- and remyelination. We found that even low doses of CDP-choline effectively enhanced early remyelination. The beneficial effects on myelin regeneration were accompanied by higher numbers of oligodendrocytes. In conclusion, CDP-choline could become a promising regenerative substance for patients with multiple sclerosis and should be tested in a clinical trial