Effects of a Novel GPR55 Antagonist on the Arachidonic Acid Cascade in LPS-Activated Primary Microglial Cells

Abstract

Neuroinflammation is a crucial process to maintain homeostasis in the central nervous system (CNS). However, chronic neuroinflammation is detrimental, and it is described in the pathogenesis of CNS disorders, including Alzheimer's disease (AD) and depression. This process is characterized by the activation of immune cells, mainly microglia. The role of the orphan G-protein-coupled receptor 55 (GPR55) in inflammation has been reported in different models. However, its role in neuroinflammation in respect to the arachidonic acid (AA) cascade in activated microglia is still lacking of comprehension. Therefore, we synthesized a novel GPR55 antagonist (KIT 10, 0.1–25 µM) and tested its effects on the AA cascade in lipopolysaccharide (LPS, 10 ng / mL)-treated primary rat microglia using Western blot and EIAs. We show here that KIT 10 potently prevented the release of prostaglandin E2 (PGE2), reduced microsomal PGE2 synthase (mPGES-1) and cyclooxygenase-2 (COX-2) synthesis, and inhibited the phosphorylation of Ikappa B-alpha (IκB-α), a crucial upstream step of the inflammation-related nuclear factor-kappaB (NF-κB) signaling pathway. However, no effects were observed on COX-1 and -2 activities and mitogen-activated kinases (MAPK). In summary, the novel GPR55 receptor antagonist KIT 10 reduces neuroinflammatory parameters in microglia by inhibiting the COX-2/PGE2 pathway. Further experiments are necessary to better elucidate its effects and mechanisms. Nevertheless, the modulation of inflammation by GPR55 might be a new therapeutic option to treat CNS disorders with a neuroinflammatory background such as AD or depression.

Targeting Oxidative Stress: Novel Coumarin-Based Inverse Agonists of GPR55

“…To confirm that the observed antioxidative effects by KIT C and KIT H are mediated via GPR55 receptors, we knocked out the GPR55 in SK-N-SH cells using a commercial CRISPR/Cas9 system and repeated the ROS-MTT assay in the knockout cell line. In line with the previous results of coumarin-based compounds [10,11], KIT C and KIT H exerted its effects by binding to GPR55, since the observed antioxidative effects of both compounds on ROS-induced cell death were abolished in GPR55-knockout cells.…”

“…It has been shown before, that coumarin-based compounds act as ligands on the GPR55 [10,11,26]. However, the receptor binding profile of the novel coumarin-based compounds KIT C and KIT H was not evaluated yet, for example by radioligand assays.…”

“…Besides the demonstrated antioxidative effects, further research on KIT C and KIT H should focus on neuroinflammatory effects of the novel coumarin-based compounds. For other coumarin-scaffolds, GPR55-dependent neuroinflammatory effects have already been shown [10,11].…”

“…Besides these widely used GPR55 ligands, coumarin-derivates show antagonistic coupled to inverse agonistic activities on GPR55-dependent neuroinflammatory processes as reported recently [10,11]. Agonistic activities at G-protein coupled receptors (GPCRs) affect downstream signal cascades activated by the receptor, such as the phosphorylation of protein kinases or the translocation of transcriptional factors in the cell's nucleus.…”

Functional Selectivity of Coumarin Derivates Acting via GPR55 in Neuroinflammation

“…Coumarin-based derivates have been shown to elicit anti-neuroinflammatory effects in primary microglia, decreasing lipopolysaccharide (LPS)-induced PGE 2 release [13,14]. High levels of PGE 2 promote brain injury and therefore act as a pro-inflammatory driver [15].…”