Identification of Sphingosine 1-Phosphate Receptor Subtype 1 (S1P(1)) as a Pathogenic Factor in Transient Focal Cerebral Ischemia

Abstract

Medically relevant roles of receptor-mediated sphingosine 1-phosphate (S1P) signaling have become a successful or promising target for multiple sclerosis or cerebral ischemia. Animal-based proof-of-concept validation for the latter is particularly through the neuroprotective efficacy of FTY720, a non-selective S1P receptor modulator, presumably via activation of S1P(1). In spite of a clear link between S1P signaling and cerebral ischemia, it remains unknown whether the role of S1P(1) is pathogenic or neuroprotective. Here, we investigated the involvement of S1P(1) along with its role in cerebral ischemia using a transient middle cerebral artery occlusion ("tMCAO") model. Brain damage following tMCAO, as assessed by brain infarction, neurological deficit score, and neural cell death, was reduced by oral administration of AUY954, a selective S1P(1) modulator as a functional antagonist, in a therapeutic paradigm, indicating that S1P(1) is a pathogenic mediator rather than a neuroprotective mediator. This pathogenic role of S1P(1) in cerebral ischemia was reaffirmed because tMCAO-induced brain damage was reduced by genetic knockdown with an intracerebroventricular microinjection of S1P(1) shRNA lentivirus into the brain. Genetic knockdown of S1P(1) or AUY954 exposure reduced microglial activation, as assessed by reduction in the number of activated microglia and reversed morphology from amoeboid to ramified, and microglial proliferation in ischemic brain. Its role in microglial activation was recapitulated in lipopolysaccharide-stimulated primary mouse microglia, in which the mRNA expression level of TNF-alpha and IL-1 beta, well-known markers for microglial activation, was reduced in microglia transfected with S1P(1) siRNA. These data suggest that the pathogenic role of S1P(1) is associated with microglial activation in ischemic brain. Additionally, the pathogenic role of S1P(1) in cerebral ischemia appears to be associated with the blood-brain barrier disruption and brain-derived neurotrophic factor (BDNF) downregulation. Overall, findings from the current study clearly identify S1P(1) signaling as a pathogenic factor in transient focal cerebral ischemia, further implicating S1P(1) antagonists including functional antagonists as plausible therapeutic agents for human stroke.