Lysophosphatidic acid receptor 1 (LPA(1)) plays critical roles in microglial activation and brain damage after transient focal cerebral ischemia

Abstract

Background Lysophosphatidic acid receptor 1 (LPA(1)) is in the spotlight because its synthetic antagonist has been under clinical trials for lung fibrosis and psoriasis. Targeting LPA(1) might also be a therapeutic strategy for cerebral ischemia because LPA(1) triggers microglial activation, a core pathogenesis in cerebral ischemia. Here, we addressed this possibility using a mouse model of transient middle cerebral artery occlusion (tMCAO). Methods To address the role of LPA(1) in the ischemic brain damage, we used AM095, a selective LPA(1) antagonist, as a pharmacological tool and lentivirus bearing a specific LPA(1) shRNA as a genetic tool. Brain injury after tMCAO challenge was accessed by determining brain infarction and neurological deficit score. Role of LPA(1) in tMCAO-induced microglial activation was ascertained by immunohistochemical analysis. Proinflammatory responses in the ischemic brain were determined by qRT-PCR and immunohistochemical analyses, which were validated in vitro using mouse primary microglia. Activation of MAPKs and PI3K/Akt was determined by Western blot analysis. Results AM095 administration immediately after reperfusion attenuated brain damage such as brain infarction and neurological deficit at 1 day after tMCAO, which was reaffirmed by LPA(1) shRNA lentivirus. AM095 administration also attenuated brain infarction and neurological deficit at 3 days after tMCAO. LPA(1) antagonism attenuated microglial activation; it reduced numbers and soma size of activated microglia, reversed their morphology into less toxic one, and reduced microglial proliferation. Additionally, LPA(1) antagonism reduced mRNA expression levels of proinflammatory cytokines and suppressed NF-kappa B activation, demonstrating its regulatory role of proinflammatory responses in the ischemic brain. Particularly, these LPA(1)-driven proinflammatory responses appeared to occur in activated microglia because NF-kappa B activation occurred mainly in activated microglia in the ischemic brain. Regulatory role of LPA(1) in proinflammatory responses of microglia was further supported by in vitro findings using lipopolysaccharide-stimulated cultured microglia, showing that suppressing LPA(1) activity reduced mRNA expression levels of proinflammatory cytokines. In the ischemic brain, LPA(1) influenced PI3K/Akt and MAPKs; suppressing LPA(1) activity decreased MAPK activation and increased Akt phosphorylation. Conclusion This study demonstrates that LPA(1) is a new etiological factor for cerebral ischemia, strongly indicating that its modulation can be a potential strategy to reduce ischemic brain damage.