Blocking S1P4 signaling attenuates brain injury in mice with ischemic stroke

• The FTY720 analog NXC736 is an S1P 4 -selective functional antagonist. • S1P 4 is a novel pathogenic factor of ischemic stroke. • The pathogenic function of S1P 4 is associated with neuroinflammation like microglial activation. • NLRP3 inflammasome activation underlies the S1P 4 -mediated pathogenesis of ischemic stroke. The functions of S1P receptors have been revealed using genetic and pharmacological tools, including the potent non-selective modulator FTY720. However, studies on subtype-specific agonists and antagonists are limited; hence, the role of S1P 4 remains unclear. To identify a novel function of S1P 4 as a pathogenic factor in stroke using a newly developed S1P 4 -selective modulator and S1P 4 knockdown. Heteroaromatic analogs of FTY720 were synthesized, a β-arrestin assay was conducted against S1P receptors, and the developed compound (NXC736) was characterized as a functional S1P 4 antagonist. To clarify the function of S1P 4 , the therapeutic potential of NXC736 in ischemic stroke was determined using a transient middle cerebral artery occlusion (tMCAO) mouse model, which was validated using S1P 4 knockdown. The S1P 4 -dependent pathogenic mechanisms were determined using immunohistochemical and biochemical analyses. Molecular modeling studies provide valuable clues for understanding S1P 4 selectivity of NXC736. NXC736 contains a triazole ring instead of a phenyl ring and exhibits S1P 4 -selective activity as a functional antagonist. Its action on S1P 4 does not require phosphorylation by sphingosine kinase 2. Notably, NXC736 exhibited substantial therapeutic activity against ischemic stroke by attenuating tMCAO-induced acute brain injuries, including brain infarction, neurological deficits, and neuronal apoptosis. This suggested that S1P 4 is a pathogenic factor in ischemic stroke. This function was confirmed using AAV-based S1P 4 knockdown. NXC736 or S1P 4 knockdown attenuated blood–brain barrier disruption, neutrophil infiltration, microglial activation and proliferation, and the upregulation of pro-inflammatory cytokines, thereby demonstrating that S1P 4 influences neuroinflammatory responses in ischemic stroke. The underlying mechanisms were activation of NLRP3 inflammasome, NF-κB, and MAPKs. S1P 4 also contributed to chronic brain injuries caused by ischemic stroke because NXC736 exerted long-term neuroprotective effects against tMCAO challenge. Using a functional S1P 4 antagonist (NXC736) and a genetic tool for S1P 4 knockdown, we identified S1P 4 as a novel pathogenic factor in ischemic stroke.

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