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Abstract
Sphingosine 1-phosphate (S1P) is a signalling lipid that mediates biological processes through five G-protein coupled receptors (S1PR1-5). S1P synthesis occurs through the phosphorylation of sphingosine by sphingosine kinase 1 (SphK1) or SphK2. S1P levels and SphK activity are reduced in multiple neurodegenerative paradigms including Alzheimer’s disease (AD). Pharmacological targeting of S1PRs is neuroprotective in animal models of these diseases.
This thesis investigates the cellular and molecular mechanisms behind S1P-mediated neuroprotection. S1P up-regulates four neurotrophic genes (BDNF, PDGFB, HBEGF, and LIF) in primary astrocytes, but not neurons. Induction of these genes is mainly driven by S1PR2 signalling, with minor contributions from S1PR1. Phosphoproteomic analysis showed time-dependent activation of canonical pathways such as the mitogen-activated protein kinase and RhoA pathways, and phosphorylation of cJUN and Yes associated protein (YAP). Immediate early genes (IEGs) were also induced with S1P. Transcription start sites of these neurotrophic factors show predicted binding sites for cJUN and YAP, and the IEGs. While distinct phosphosites were regulated by S1PR1 and S1PR2 signalling, a large subset was regulated by both. However, the RhoA-YAP pathway was exclusively activated by S1PR2.
The clinical drug Fingolimod (FTY720) is a sphingosine analogue that is phosphorylated in vivo by SphK2 to form the S1P structural analogue FTY720-P. Previous reports detailing the neuroprotective properties of S1PRs used FTY720-P to investigate the neurotrophic response. Unlike S1P, FTY720-P does not activate the S1PR2-RhoA-YAP pathway in astrocytes. Accordingly, S1P was a much more potent inducer of IEGs and neurotrophic gene expression than FTY720-P. Additionally, S1P but not FTY720-P significantly attenuated excitotoxic neuronal cell death in vitro when co-cultured with glia. Neuroprotection was ablated upon incubation with a LIF neutralising antibody, but not with antagonists to BDNF or HBEGF signalling.
I have therefore established a novel neuroprotective pathway in which S1P stimulates secretion of the growth factor LIF by astrocytes, which protects neurons against excitotoxic cell death. This pathway requires dual S1PR1 and S1PR2 signalling in astrocytes, thus explaining the inefficacy of FTY720-P. Current S1PR therapies do not target S1PR2 and targeting this receptor may be a novel therapeutic option for AD and other neurodegenerative conditions.