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. In spite of a clear link between S1P signaling and cerebral ischemia, it remains unknown whether the role of S1P is pathogenic or neuroprotective. Here, we investigated the involvement of S1P 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 modulator as a functional antagonist, in a therapeutic paradigm, indicating that S1P is a pathogenic mediator rather than a neuroprotective mediator. This pathogenic role of S1P in cerebral ischemia was reaffirmed because tMCAO-induced brain damage was reduced by genetic knockdown with an intracerebroventricular microinjection of S1P shRNA lentivirus into the brain. Genetic knockdown of S1P 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-α and IL-1β, well-known markers for microglial activation, was reduced in microglia transfected with S1P siRNA. These data suggest that the pathogenic role of S1P is associated with microglial activation in ischemic brain. Additionally, the pathogenic role of S1P 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 signaling as a pathogenic factor in transient focal cerebral ischemia, further implicating S1P antagonists including functional antagonists as plausible therapeutic agents for human stroke.

The signaling axis of glucagon-like peptide-1 (GLP-1)/GLP-1 receptor (GLP-1R) has been an important component in overcoming diabetes, and recent reports have uncovered novel beneficial roles of this signaling axis in central nervous system (CNS) disorders, such as Alzheimer's disease, Parkinson's disease, and cerebral ischemia, accelerating processes for exendin-4 repositioning. Here, we studied whether multiple sclerosis (MS) could be a complement to the CNS disorders that are associated with the GLP-1/GLP-1R signaling axis. Both components of the signaling axis, GLP-1 and GLP-1R proteins, are expressed in neurons, astrocytes, and microglia in the spinal cord of normal mice. In particular, they are abundant in Iba1-positive microglia. Upon challenge by experimental autoimmune encephalomyelitis (EAE), an animal model of MS, the mRNA expression of both GLP-1 and GLP-1R was markedly downregulated in EAE-symptomatic spinal cords, indicating attenuated activity of GLP-1/GLP-1R signaling in EAE. Such a downregulation obviously occurred in LPS-stimulated rat primary microglia, a main cell type to express both GLP-1 and GLP-1R, further indicating attenuated activity of GLP-1/GLP-1R signaling in activated microglia. To investigate whether increased activity of GLP-1R has a therapeutic benefit, exendin-4 (5 μg/kg, i.p.), a GLP-1R agonist, was administered daily to EAE-symptomatic mice. Exendin-4 administration to symptomatic EAE mice significantly improved the clinical signs of the disease, along with the reversal of histopathological sequelae such as cell accumulation, demyelination, astrogliosis, microglial activation, and morphological transformation of activated microglia in the injured spinal cord. Such an improvement by exendin-4 was comparable to that by FTY720 (3 mg/kg, i.p.), a drug for MS. The neuroprotective effects of exendin-4 against EAE were also associated with decreased mRNA expression of proinflammatory cytokines, such as interleukin (IL)-17, IL-1β, IL-6, and tumor necrosis factor (TNF)-α, all of which are usually upregulated in injured sites of the EAE spinal cord. Interestingly, exendin-4 exposure similarly reduced mRNA levels of IL-1β and TNF-α in LPS-stimulated microglia. Furthermore, exendin-4 administration significantly attenuated activation of NF-κB signaling in EAE spinal cord and LPS-stimulated microglia. Collectively, the current study demonstrates the therapeutic potential of exendin-4 for MS by reducing immune responses in the CNS, highlighting the importance of the GLP-1/GLP-1R signaling axis in the development of a novel therapeutic strategy for MS.

Sphingosine 1-phosphate (S1P) signaling has emerged as a drug target in cerebral ischemia. Among S1P receptors, S1P 2 was recently identified to mediate ischemic brain injury. But, pathogenic mechanisms are not fully identified, particularly in view of microglial activation, a core pathogenesis in cerebral ischemia. Here, we addressed whether microglial activation is the pathogenesis of S1P 2 -mediated brain injury in mice challenged with transient middle cerebral artery occlusion (tMCAO). To suppress S1P 2 activity, its specific antagonist, JTE013 was given orally to mice immediately after reperfusion. JTE013 administration reduced the number of activated microglia and reversed their morphology from amoeboid to ramified microglia in post-ischemic brain after tMCAO challenge, along with attenuated microglial proliferation. Moreover, JTE013 administration attenuated M1 polarization in post-ischemic brain. This S1P 2 -directed M1 polarization appeared to occur in activated microglia, which was evidenced upon JTE013 exposure in vivo as suppressed M1-relevant NF-κB activation in activated microglia of post-ischemic brain. Moreover, JTE013 exposure or S1P 2 knockdown reduced expression levels of M1 markers in vitro in lipopolysaccharide-driven M1 microglia. Additionally, suppressing S1P 2 activity attenuated activation of M1-relevant ERK1/2 and JNK in post-ischemic brain or lipopolysaccharide-driven M1 microglia. Overall, our study demonstrated that S1P 2 regulated microglial activation and M1 polarization in post-ischemic brain.

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-κB 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-κB 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 indicatin...

The pathogenesis of psoriasis, an immune-mediated chronic skin barrier disease, is not fully understood yet. Here, we identified lysophosphatidic acid (LPA) receptor 5 (LPA5)-mediated signaling as a novel pathogenic factor in psoriasis using an imiquimod-induced psoriasis mouse model. Amounts of most LPA species were markedly elevated in injured skin of psoriasis mice, along with LPA5 upregulation in injured skin. Suppressing the activity of LPA5 with TCLPA5, a selective LPA5 antagonist, improved psoriasis symptoms, including ear thickening, skin erythema, and skin scaling in imiquimod-challenged mice. TCLPA5 administration attenuated dermal infiltration of macrophages that were found as the major cell type for LPA5 upregulation in psoriasis lesions. Notably, TCLPA5 administration attenuated the upregulation of macrophage NLRP3 in injured skin of mice with imiquimod-induced psoriasis. This critical role of LPA5 in macrophage NLRP3 was further addressed using lipopolysaccharide-primed bone marrow-derived macrophages. LPA exposure activated NLRP3 inflammasome in lipopolysaccharide-primed cells, which was evidenced by NLRP3 upregulation, caspase-1 activation, and IL-1β maturation/secretion. This LPA-driven NLRP3 inflammasome activation in lipopolysaccharide-primed cells was significantly attenuated upon LPA5 knockdown. Overall, our findings establish a pathogenic role of LPA5 in psoriasis along with an underlying mechanism, further suggesting LPA5 antagonism as a potential strategy to treat psoriasis.