Investigating the role of fractalkine an S1P receptors on oxidative stress in the CNS

Abstract

Scientific Abstract: The fractalkine ligand (CX3CL1) is a unique chemokine that is synthesised as a transmembrane protein. One of the unique features of fractalkine is its ability to exist as both a membrane tethered adhesion molecule and as a soluble chemotactic ligand via activation of its G protein-coupled receptor CX3CR1. Importantly, the fractalkine ligand is expressed in astrocytes and reported to be involved in regulating the inflammatory process. Here, we examined how fractalkine is regulated and cleaved from human astrocytes. Our studies show, for the first time, that ADAM10 is the main protease responsible for the cleavage of fractalkine from the surface of astrocytes under pro-inflammatory conditions. We also demonstrate the involvement of p38 MAPK and NF-κB in this regulation of sCX3CL1 (Results, Chapter 3). Using the S1PR agonist, pFTY720 and recombinant fractalkine (rCX3CL1), we investigated the effects of S1PR and CX3CR1 activation on oxidative stress induced demyelination in organotypic cerebellar slice cultures treated with bolus concentrations of H2O2 (0.1-1mM) and low-continuous H2O2 (~20μM) generated from glucose oxidase and catalase (GOX-CAT). We report that pFTY720 and fractalkine attenuated both bolus and continuous H2O2-induced demyelination (Results, chapter 4 and 5). In addition, both bolus H2O2 and GOX-CAT treatments caused a significant decrease in vimentin fluorescence in these slice culture models with no change observed in microglial Iba1 fluorescence. Importantly, pre-treatment with pFTY720 partially attenuated bolus H2O2-induced decrease in vimentin fluorescence, while significantly attenuating GOX-CAT induced decrease in vimentin fluorescence. Of note, pFTY720 did not reduce GOX-CAT induced levels of the inflammatory cytokines, IL-6 and fractalkine (Results, chapter 4). In contrast, we observed that rCX3CL1 attenuated bolus H2O2-induced decrease in vimentin fluorescence but had no effect on GOX-CAT induced decrease in vimentin fluorescence. Again we observed that rCX3CL1 did not reduce GOX-CAT induced levels of IL-6 nor did it alter Iba1 fluorescence (Results, chapter 5). Overall, this study establishes a new model of oxidative stress induced demyelination through the use of GOX-CAT enzymes and suggests that pFTY720 can attenuate oxidative stress induced demyelination. We also highlight the potential for fractalkine receptor modulation as a new therapeutic avenue for demyelinating diseases. Lay Abstract The brain is made up of four major types of brain cells. Nerve cells (neurons) are specialised cells that process and carry ‘messages’ through electrical signals. Oligodendrocytes are cells which are wrapped around neurons and form a layer of insulation (myelin), which help carry the neuron ‘message’. Microglia, are cells which form the brains immune system. Astrocytes help to form and maintain the network on which neurons grow. All brain cells use oxygen to make energy (metabolism) and bi-products of this reaction, such as hydrogen peroxide (H2O2), are created for a short period of time. The brain normally clears and detoxifies these bi-products, before they become harmful as they can cause oxidative damage and inflammation. All brain cells have a large number of proteins on their surface called receptors, which are used to communicate with other cells. These receptors are activated by molecules released by other cells and can also be activated by drugs. In this thesis we looked at the drug that is used to treat multiple sclerosis (MS) called Gilenya (pFTY720). This drug can bind specific receptors, which are called sphingosine 1-phosphate receptors (S1PRs) on brain cells and immune cells. T cells are one type of immune cell that are thought to cause damage in the brain during MS. FTY720 is thought to prevent these T cells from entering the brain and causing damage to nerve cells, which can lead to the symptoms of MS. We also looked at a molecule called fractalkine, which is naturally released from neurons and astrocytes. Fractalkine can activate its own receptor called CX3CR1. In many brain diseases such as MS and Alzheimer’s disease cells start to die from the build up of toxic products such as H2O2. In this thesis we used cultures of astrocytes and brain slices from mice to investigate the drug Gilenya and the natural molecule fractalkine. We showed that they were protective of cells from toxic levels of H2O2. This research may provide a better understanding of how we can modulate different brain cell functions to develop new drugs for brain-related illnesses.