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Piezo1 is a mechanosensitive ion channel that facilitates the translation of extracellular mechanical cues to intracellular molecular signaling cascades through a process termed, mechanotransduction. In the central nervous system (CNS), mechanically gated ion channels are important regulators of neurodevelopmental processes such as axon guidance, neural stem cell differentiation, and myelination of axons by oligodendrocytes. Here, we present evidence that pharmacologically mediated overactivation of Piezo1 channels negatively regulates CNS myelination. Moreover, we found that the peptide GsMTx4, an antagonist of mechanosensitive cation channels such as Piezo1, is neuroprotective and prevents chemically induced demyelination. In contrast, the positive modulator of Piezo1 channel opening, Yoda-1, induces demyelination and neuronal damage. Using an ex vivo murine-derived organotypic cerebellar slice culture model, we demonstrate that GsMTx4 attenuates demyelination induced by the cytotoxic lipid, psychosine. Importantly, we confirmed the potential therapeutic effects of GsMTx4 peptide in vivo by co-administering it with lysophosphatidylcholine (LPC), via stereotactic injection, into the cerebral cortex of adult mice. GsMTx4 prevented both demyelination and neuronal damage usually caused by the intracortical injection of LPC in vivo; a well-characterized model of focal demyelination. GsMTx4 also attenuated both LPC-induced astrocyte toxicity and microglial reactivity within the lesion core. Overall, our data suggest that pharmacological activation of Piezo1 channels induces demyelination and that inhibition of mechanosensitive channels, using GsMTx4, may alleviate the secondary progressive neurodegeneration often present in the latter stages of demyelinating diseases., (© 2019 Wiley Periodicals, Inc.)

Astrocytes are important for information processing in the brain and they achieve this by fine-tuning neuronal communication via continuous uptake and release of biochemical modulators of neurotransmission and synaptic plasticity. Often overlooked are their important functions in mechanosensation. Indeed, astrocytes can detect pathophysiological changes in the mechanical properties of injured, ageing, or degenerating brain tissue. We have recently shown that astrocytes surrounding mechanically-stiff amyloid plaques upregulate the mechanosensitive ion channel, Piezo1. Moreover, ageing transgenic Alzheimer's rats harboring a chronic peripheral bacterial infection displayed enhanced Piezo1 expression in amyloid plaque-reactive astrocytes of the hippocampus and cerebral cortex. Here, we have shown that the bacterial endotoxin, lipopolysaccharide (LPS), also upregulates Piezo1 in primary mouse cortical astrocyte cultures in vitro. Activation of Piezo1, via the small molecule agonist Yoda1, enhanced Ca 2+ influx in both control and LPS-stimulated astrocytes. Moreover, Yoda1 augmented intracellular Ca 2+ oscillations but decreased subsequent Ca 2+ influx in response to adenosine triphosphate (ATP) stimulation. Neither blocking nor activating Piezo1 affected cell viability. However, LPS-stimulated astrocyte cultures exposed to the Piezo1 activator, Yoda1, migrated significantly slower than reactive astrocytes treated with the mechanosensitive channel-blocking peptide, GsMTx4. Furthermore, our data show that activating Piezo1 channels inhibits the release of cytokines and chemokines, such as IL-1β, TNFα, and fractalkine (CX 3 CL1), from LPS-stimulated astrocyte cultures. Taken together, our results suggest that astrocytic Piezo1 upregulation may act to dampen neuroinflammation and could be a useful drug target for neuroinflammatory disorders of the brain., (© 2019 Wiley Periodicals, Inc.)

The endogenous oxysterol 7α, 25-dihydroxycholesterol (7α25HC) ligand activates the G protein-coupled receptor EBI2 to regulate T cell-dependant antibody response and B cell migration. We have demonstrated that EBI2 is expressed in human and mouse astrocytes, that 7α25HC induces intracellular signalling and astrocyte migration, and that EBI2 plays a role in the crosstalk between astrocytes and macrophages. Recently, we demonstrate that EBI2 regulates myelin development and inhibits LPC-induced demyelination. Here, we show that 7α25HC inhibits LPS- and IL17/TNF-induced pro-inflammatory cytokine release in astrocytes. We observe the following: 1. Human astrocytes treated with IL17/TNF increases the nuclear translocation of NFκB, which is attenuated by pre-treatment with 7α25HC; 2. IL17/TNF increases cell impedance in human astrocytes, which is also attenuated by pre-treatment with 7α25HC; 3. The EBI2 antagonist NIBR189 inhibits these effects of 7α25HC, supporting the role of EBI2; 4. in vivo data corroborate these in vitro findings, showing that EBI2 knock-out (KO) animals display enhanced pro-inflammatory cytokine in response to LPS challenge, in the brain. These results demonstrate a role for oxysterol/EBI2 signalling in attenuating the response of astrocytes to pro-inflammatory signals as well as limiting the levels of pro-inflammatory cytokines in the brain., (Copyright © 2018 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Sphingosine 1-phosphate receptors (S1PR) are G protein-coupled and compose a family with five subtypes, S1P1R-S1P5R. The drug Gilenya ® (Novartis, Basel, Switzerland) (Fingolimod; FTY720) targets S1PRs and was the first oral therapy for patients with relapsing-remitting multiple sclerosis (MS). The phosphorylated form of FTY720 (pFTY720) binds S1PRs causing initial agonism, then subsequent receptor internalization and functional antagonism. Internalization of S1P1R attenuates sphingosine 1-phosphate (S1P)-mediated egress of lymphocytes from lymph nodes, limiting aberrant immune function in MS. pFTY720 also exerts direct actions on neurons and glial cells which express S1PRs. In this study, we investigated the regulation of pro-inflammatory chemokine release by S1PRs in enriched astrocytes and microglial cultures. Astrocytes and microglia were stimulated with lipopolysaccharide (LPS) and increases in C-X-C motif chemokine 5 (CXCL5), also known as LIX (lipopolysaccharide-induced CXC chemokine) expression were quantified. Results showed that pFTY720 attenuated LPS-induced CXCL5 (LIX) protein release from astrocytes, as did the S1P1R selective agonist, SEW2871. In addition, pFTY720 blocked messenger ribonucleic acid (mRNA) transcription of the chemokines, (i) CXCL5/LIX, (ii) C-X-C motif chemokine 10 (CXCL10) also known as interferon gamma-induced protein 10 (IP10) and (iii) chemokine (C-C motif) ligand 2 (CCL2) also known as monocyte chemoattractant protein 1 (MCP1). Interestingly, inhibition of sphingosine kinase attenuated LPS-induced increases in mRNA levels of all three chemokines, suggesting that LPS-TLR4 (Toll-like receptor 4) signalling may enhance chemokine expression via S1P-S1PR transactivation. Lastly, these observations were not limited to astrocytes since we also found that pFTY720 attenuated LPS-induced release of CXCL5 from microglia. These data highlight a role for S1PR signalling in regulating the levels of chemokines in glial cells and support the notion that pFTY720 efficacy in multiple sclerosis may involve the direct modulation of astrocytes and microglia., (© 2018 International Society for Neurochemistry.)

Krabbe disease is a fatal rare inherited lipid storage disorder affecting 1:100,000 births. This illness is caused by mutations in the galc gene encoding for the enzyme galactosylceramidase (GALC). Dysfunction of GALC has been linked to the toxic build-up of the galactolipid, galactosylsphingosine (psychosine), which induces cell death of oligodendrocytes. Previous studies show that phospholipase A2 (PLA2) may play a role in psychosine induce cell death. Here, we demonstrate that non-selective inhibition of cPLA2/sPLA2 and selective inhibition of cPLA2, but not sPLA2, also attenuates psychosine-induced cell death of human astrocytes. This study shows that extracellular calcium is required for psychosine induced cell death, but intracellular calcium release, reactive oxygen species or release of soluble factors are not involved. These findings suggest a cell autonomous effect, at least in human astrocytes. Supporting a role for PLA2 in psychosine-induced cell death of oligodendrocytes and astrocytes, the results show inhibition of PLA2 attenuates psychosine-induced decrease in the expression of astrocyte marker vimentin as well as myelin basic protein (MBP), myelin oligodendrocyte glycoprotein (MOG) and the neuronal marker SMI-32 in organotypic slice cultures. These findings provide further mechanistic details of psychosine-induced death of glia and suggest a role for PLA2 in the process. This work also supports the proposal that novel drugs for Krabbe disease may require testing on astrocytes as well as oligodendrocytes for more holistic prediction of pre-clinical and clinical efficacy.

Background: The fractalkine (CX3CR1) ligand is expressed in astrocytes and reported to be neuroprotective. When cleaved from the membrane, soluble fractalkine (sCX3CL1) activates the receptor CX3CR1. Although somewhat controversial, CX3CR1 is reported to be expressed in neurons and microglia. The membrane-bound form of CX3CL1 additionally acts as an adhesion molecule for microglia and infiltrating white blood cells. Much research has been done on the role of fractalkine in neuronal cells; however, little is known about the regulation of the CX3CL1 ligand in astrocytes., Methods: The mechanisms involved in the up-regulation and cleavage of CX3CL1 from human astrocytes were investigated using immunocytochemistry, Q-PCR and ELISA. All statistical analysis was performed using GraphPad Prism 5., Results: A combination of ADAM17 (TACE) and ADAM10 protease inhibitors was found to attenuate IL-1β-, TNF-α- and IFN-γ-induced sCX3CL1 levels in astrocytes. A specific ADAM10 (but not ADAM17) inhibitor also attenuated these effects, suggesting ADAM10 proteases induce release of sCX3CL1 from stimulated human astrocytes. A p38 MAPK inhibitor also attenuated the levels of sCX3CL1 upon treatment with IL-1β, TNF-α or IFN-γ. In addition, an IKKβ inhibitor significantly reduced the levels of sCX3CL1 induced by IL-1β or TNF-α in a concentration-dependent manner, suggesting a role for the NF-kB pathway., Conclusions: In conclusion, this study shows that the release of soluble astrocytic fractalkine is regulated by ADAM10 proteases with p38 MAPK also playing a role in the fractalkine shedding event. These findings are important for understanding the role of CX3CL1 in healthy and stimulated astrocytes and may benefit our understanding of this pathway in neuro-inflammatory and neurodegenerative diseases.

EBI2 is a G protein-coupled receptor activated by oxysterol 7α, 25-dihydroxycholesterol (7α25HC) and regulates T cell-dependant antibody response and B cell migration. We recently found EBI2 is expressed in human astrocytes, regulates intracellular signalling and modulates astrocyte migration. Here, we report that LPS treatment of mouse astrocytes alters mRNA levels of EBI2 and oxysterols suggesting that the EBI2 signalling pathway is sensitive to LPS-mediated immune challenge. We also find that conditioned media obtained from LPS-stimulated mouse astrocytes induces macrophage migration, which is inhibited by the EBI2 antagonist NIBR189. These results demonstrate a role for the EBI2 signalling pathway in astrocytes as a sensor for immune challenge and for communication with innate immune cells such as macrophages.

Background: BAF312 (Siponimod) is a dual agonist at the sphingosine-1 phosphate receptors, S1PR1 and S1PR5. This drug is currently undergoing clinical trials for the treatment of secondary progressive multiple sclerosis (MS). Here, we investigated the effects of BAF312 on isolated astrocyte and microglia cultures as well as in slice culture models of demyelination., Methods: Mouse and human astrocytes were treated with S1PR modulators and changes in the levels of pERK, pAkt, and calcium signalling as well as S1PR1 internalization and cytokine levels was investigated using Western blotting, immunochemistry, ELISA and confocal microscopy. Organotypic slice cultures were prepared from the cerebellum of 10-day-old mice and treated with lysophosphatidylcholine (LPC), psychosine and/or S1PR modulators, and changes in myelination states were measured by fluorescence of myelin basic protein and neurofilament H., Results: BAF312 treatment of human and mouse astrocytes activated pERK, pAKT and Ca(2+) signalling as well as inducing S1PR1 internalization. Notably, activation of S1PR1 increased pERK and pAKT in mouse astrocytes while both S1PR1 and S1PR3 equally increased pERK and pAKT in human astrocytes, suggesting that the coupling of S1PR1 and S1PR3 to pERK and pAKT differ in mouse and human astrocytes. We also observed that BAF312 moderately attenuated lipopolysaccharide (LPS)- or TNFα/IL17-induced levels of IL6 in both astrocyte and microglia cell cultures. In organotypic slice cultures, BAF312 reduced LPC-induced levels of IL6 and attenuated LPC-mediated demyelination. We have shown previously that the toxic lipid metabolite psychosine induces demyelination in organotypic slice cultures, without altering the levels of cytokines, such as IL6. Importantly, psychosine-induced demyelination was also attenuated by BAF312., Conclusions: Overall, this study suggests that BAF312 can modulate glial cell function and attenuate demyelination, highlighting this drug as a further potential therapy in demyelinating disorders, beyond MS.

The G protein-coupled receptor EBI2 (Epstein-Barr virus-induced gene 2) is activated by 7α, 25-dihydroxycholesterol (7α25HC) and plays a role in T cell-dependant antibody response and B cell migration. Aberrant EBI2 signaling is implicated in a range of autoimmune disorders however its role in the CNS remains unknown. Here we characterize the functional role of EBI2 in GLIA cells using primary human astrocytes and EBI2 knockout animals. We find human and mouse astrocytes express EBI2 and the enzymes necessary for synthesis and degradation of 7α25HC. In astrocytes, EBI2 activation stimulates ERK phosphorylation, Ca(2+) signaling and induces cellular migration. These results, for the first time, demonstrate a role for EBI2 in astrocyte function and suggest that modulation of this receptor may be beneficial in neuroinflammatory disorders., (© 2014 Wiley Periodicals, Inc.)

The family of interleukin 17 receptors (IL17Rs), subtypes IL17RA-IL17RE, is targeted by the group of pro-inflammatory IL17 cytokines (IL17A-F) and moreover the newly developed anti-IL17A antibody secukinumab (AIN457) has shown promise in Phase II trials in multiple sclerosis. Here, we show that human astrocytes, isolated from a fetal cerebral cortex, express IL17RA and IL17RC and in vitro treatment with IL17A increases protein levels of IL6 in human astrocytes, which is enhanced in the presence of TNFα, as determined by homogeneous time resolved fluorescence. Studies on acutely isolated mouse astrocytes are comparable to human astrocytes although the protein levels of IL6 are lower in mouse astrocytes, which also show a lower response to IL17F and IL1β in promoting IL6 levels. In human astrocytes, IL17A and TNFα also induce mRNA expression of IL6, IL8 and the Th17 cytokines CXCL1, CXCL2, and CCL20, with little effect on Th1 cytokines CXCL9, CXCL10, and CXCL11. The effects of IL17A are associated with nuclear translocation of the NF-κB transcription factor, as determined by immunocytochemistry, where treatment of human astrocytes with the inhibitors of the NF-κB pathway and with secukinumab inhibits the IL17A and IL17A/TNFα-induced increase in nuclear translocation of NF-κB and levels of IL6. Taken together the data shows that IL17A signaling plays a key role in regulating the levels of cytokines, such as IL6, in human astrocytes via a mechanism that involves NF-κB signaling and that selective inhibition of IL17A signaling attenuates levels of pro-inflammatory molecules in astrocytes.

It is well established that immunosurveillance is active in the CNS and plays a key role in several CNS disorders but the exact role of immune cells remains elusive. Thus, in the present study we investigated whether lymphocytes are protective/detrimental in in vitro models of excitotoxicty. Kainate (KA)-induced neuronal death was significantly reduced following exposure to mixed lymphocytes or purified T lymphocytes containing either activated or non-activated T-lymphocytes. Conditioned media from lymphocyte preparations, but not boiled conditioned media, was protective against KA-induced toxicity indicating soluble mediators underlie the observed neuroprotection with cytokine arrays indicating IL-16 as the likely candidate. A role for astrocytes was established as the neuroprotection was abolished in the presence of the glial toxin, fluoroacetate. Furthermore, lymphocytes inhibited p38 MAPK and ERK signalling pathways with pharmacological inhibition of these pathways mimicking the protective effect of lymphocytes. Similarly, lymphocytes were neuroprotective against oxygen-glucose deprivation (OGD)-induced cell death with the inhibition of p38 MAPK and ERK signalling pathways involved. These data indicate that lymphocytes are neuroprotective under our experimental conditions and we suggest that astrocytic activation and inhibition of MAPK signalling cascades are involved but further studies are required to investigate whether similar mechanisms underlie the actions of lymphocytes in in vivo experimental models of disease. This article is part of the Special Issue entitled 'The Synaptic Basis of Neurodegenerative Disorders'., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Background and Purpose: The sphingosine 1-phosphate receptor subtype 1 (S1P1R) is modulated by phosphorylated FTY720 (pFTY720), which causes S1P1R internalization preventing lymphocyte migration thus limiting autoimmune response. Studies indicate that internalized S1P1Rs continue to signal, maintaining an inhibition of cAMP, thus raising question whether the effects of pFTY720 are due to transient initial agonism, functional antagonism and/or continued signalling. To further investigate this, the current study first determined if continued S1P1R activation is pathway specific., Experimental Approach: Using human and rat astrocyte cultures, the effects of S1P1R activation on cAMP, pERK and Ca(2+) signalling was investigated. In addition, to examine the role of S1P1R redistribution on these events, a novel biologic (MNP301) that prevented pFTY720-mediated S1P1R redistribution was engineered., Key Results: The data showed that pFTY720 induced long-lasting S1P1R redistribution and continued cAMP signalling in rat astrocytes. In contrast, pFTY720 induced a transient increase of Ca(2+) in astrocytes and subsequent antagonism of Ca(2+) signalling. Notably, while leaving pFTY720-induced cAMP signalling intact, the novel MNP301 peptide attenuated S1P1R-mediated Ca(2+) and pERK signalling in cultured rat astrocytes., Conclusions and Implications: These findings suggested that pFTY720 causes continued cAMP signalling that is not dependent on S1P1R redistribution and induces functional antagonism of Ca(2+) signalling after transient stimulation. To our knowledge, this is the first report demonstrating that pFTY720 causes continued signalling in one pathway (cAMP) versus functional antagonism of another pathway (Ca(2+)) and which also suggests that redistributed S1P1Rs may have differing signalling properties from those expressed at the surface., (© 2013 The British Pharmacological Society.)

Sphingosine-1-phosphate (S1P) receptors are widely expressed in the central nervous system where they are thought to regulate glia cell function. The phosphorylated version of fingolimod/FTY720 (FTY720P) is active on a broad spectrum of S1P receptors and the parent compound is currently in phase III clinical trials for the treatment of multiple sclerosis. Here, we aimed to identify which cell type(s) and S1P receptor(s) of the central nervous system are targeted by FTY720P. Using calcium imaging in mixed cultures from embryonic rat cortex we show that astrocytes are the major cell type responsive to FTY720P in this assay. In enriched astrocyte cultures, we detect expression of S1P1 and S1P3 receptors and demonstrate that FTY720P activates Gi protein-mediated signaling cascades. We also show that FTY720P as well as the S1P1-selective agonist SEW2871 stimulate astrocyte migration. The data indicate that FTY720P exerts its effects on astrocytes predominantly via the activation of S1P1 receptors, whereas S1P signals through both S1P1 and S1P3 receptors. We suggest that this distinct pharmacological profile of FTY720P, compared with S1P, could play a role in the therapeutic effects of FTY720 in multiple sclerosis.

Sphingosine-1-phosphate receptors (S1P1-5) are activated by the endogenous agonist S1P and are expressed in the central nervous system. In astrocytes, activation of S1P receptors leads to phosphorylation of extracellular-signal regulated kinase (ERK), a signaling cascade which plays intimate roles in cell proliferation. Fingolimod (FTY720) is in phase III clinical trials for the treatment of multiple sclerosis and its phosphorylated version (FTY720P) activates S1P receptors. We examined the effects of FTY720P on ERK phosphorylation and determined which S1P receptor subtype(s) mediated this signaling event. FTY720P augmented ERK phosphorylation in cortical cultures prepared from embryonic day 18 rat brains and was blocked by an MEK inhibitor or by pertussis toxin. Co-localisation of phosphorylated ERK occurred in glial fibrillary acidic protein (GFAP) positive astrocytes but not neurons or oligodendrocytes. Furthermore, FTY720P stimulated ERK phosphorylation in highly enriched astrocyte cultures made from postnatal day 2 rat cortices. The effects of FTY720P were mimicked by selective S1P1 receptor agonists and blocked by S1P1 receptor antagonists. Collectively, these results demonstrate that FTY720P mediates ERK phosphorylation in astrocytes via the activation of S1P1 receptors.