Alterations In Lipid Metabolism In The Nervous System By Ethanol

National Institute On Alcohol Abuse And Alcoholism

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Abstract

We have previously established that DHA metabolism to N-docosahexaenoylethanolamine (synaptamide) is a significant endogenous mechanism for promoting neurogenesis, neuritogenesis, synaptogenesis and anti-inflammatory action in a cAMP dependent manner. We further demonstrated that orphan G-protein coupled receptor 110 (GPR110, ADGRF1) is the synaptamide target receptor, triggering cAMP production with low nM potency. GPR110 belongs to adhesion GPCRs (aGPCRs), a newly emerging class of GPCR, and we discovered that synaptamide is the only small-molecule endogenous ligand thus far identified for aGPCR. We also found a specific binding of synaptamide to the GAIN domain, which in turn induces conformational changes in the intracellular regions of GPR110 and triggers G-protein activation and b-arrestin binding, providing a new framework for understanding the physiological function of aGPCRs and therapeutic targeting in the GAIN domain. During this period, we continued our investigation of the in vivo significance of this mechanism in injury/inflammation models along with characterization of the signaling pathways for the newly deorphanized GPR110. We have previously found that DHA-derived synaptamide is a potent suppressor of neuroinflammation in an LPS-induced model, by enhancing cAMP/PKA signaling and inhibiting NF-kB activation through GPR110 activation in both brain and periphery. We have also established that synaptamide can reduce the impairing effect of chronic ethanol exposure on neuronal differentiation of NSCs by counter-affecting shared targets in GPCR/cAMP signaling, offering a possible avenue for ameliorating the adverse impact of fetal alcohol exposure on neurodevelopment. During this review period, we examined the interaction between the effects of ethanol and synaptamide on neuroinflammation in vivo, as well as in vitro using primary microglia. Binge ethanol administration at a dose of (3 g/kg) enhanced LPS (1 mg/kg) induced neuroinflammation in vivo. The administration of synaptamide significantly suppressed in a GPR110-dependent manner the expression of proinflammatory indicator genes including TNF, IL-6, IL-1b and NLRP3 that were induced by LPS and further elevated with prior exposure to ethanol. The LPS-induced NLRP3 expression was dramatically increased in the ethanol-fed GPR110 KO animals compared to the ethanol-fed WT, suggesting that inflammatory responses enhanced by ethanol is further sensitized in the absence of GPR110. The ameliorative effect of synaptamide was GPR110- and cAMP-dependent. As the cAMP level is tightly regulated and balanced by adenylylcyclases (AC) and phosphodiesterases (PDE), we evaluated the effect of ethanol and synaptamide on the expression of different isoforms of AC and PDE enzymes. A binge exposure to ethanol and LPS injection significantly decreased the expression of specifically the AC8 isoform in the brain. Conversely, both ethanol and LPS injection specifically increased the expression of the PDE4b isoform. Synaptamide reversed the reduction of AC8 and normalized PDE4b, but these effects were not observed in the absence of GPR110. The suppression of PDE4 upregulation by synaptamide was also GPR110-dependent, indicating a role of GPR110 in controlling the PDE4 system. Indeed, upregulation of PDE4 by LPS and ethanol was significantly exaggerated in GPR110 KO mice. Our data indicated an adverse impact of ethanol on neuroinflammatory responses by downregulating the cAMP system. In contrast, synaptamide GPR110-dependently upregulates the cAMP system by at least partly counteracting the effect of ethanol and LPS on AC8 and PDE4 expression. The protective role of GPR10 activation by synaptamide might be attributed through the modulation of adenylate cyclase 8 and pde4b. These findings suggest that GPR110 is a novel therapeutic target to ameliorate inflammation in the brain as well as periphery, and to prevent the ethanol-induced disturbance of the innate immune system. To further characterize the biological processes associated with GPR110, we probed GPR110-interacting proteins by in-cell cross-linking, co-immunoprecipitation and quantitative mass spectrometry. Adhesion G protein-coupled receptors (aGPCRs/ADGRs) including GPR110 have a functional role in cell adhesion besides cell signaling. While the intracellular domain of ADGRs is coupled to G-proteins, -arrestin or other scaffolding proteins to initiate signaling cascades, the characteristically large extracellular domain of ADGRs contains adhesion modules that interact with the extracellular matrix or cell surface proteins. We identified potential GPR110 interacting proteins including occluding, a tight junction protein necessary for paracellular barrier formation, cell polarization, differentiation and proliferation, as well as regulation of progenitor self-renewal and survival in developing cortex. The interaction of occludin with GPR110 was five times less in a functionally inactive mutant form of GPR110 (F663S) which was shown to be associated with schizophrenia in a Swedish exome-sequencing data set. We also found that the F663S mutant fails to localize at the plasma membrane. Because occludin regulates the tight junction paracellular permeability, we assessed the role of the newly identified GPR110-occludin interaction on blood-brain barrier (BBB) permeability. We intravenously injected a small non-permeable fluorescent dye (Rhodamine 800) into the tail vein of GPR110 KO or WT mice and examined its penetration into the brain by near infrared whole brain imaging. While the fluorescence signal was negligible in the WT brain, the signal was significantly enhanced in the GPR110 KO brain, indicating compromised BBB function in the KO mice. Our data suggest that the GPR110 interaction with a tight junction protein occludin may play an important role in regulating the BBB permeability. Further characterization of the potential interacting partners of GPR110 identified in this study will enhance our understanding of the physiologic and pathologic function of GPR110. We continued to investigate the role of GPR110 activation using human induced pluripotent stem cells (iPSCs) that we initiated during the last review period. GPR110 is expressed in iPSCs and neural progenitor cells (NPCs) differentiated from iPSCs, and GPR110 ligands dose-dependently increased cAMP as we observed in mouse neural stem cells. While neuronal cells retained GPR110 expression the GFAP-positive astrocytes do not show GPR110 expression. GPR110 ligands promoted neurite growth, accelerated spontaneous activity and synchrony, and increased the expression of TBR1, a transcription factor critical for controlling glutamatergic neuronal cell fate, in human iPSC-derived cortical neurons. The GPR110 signaling and its developmental function recapitulated in human iPSC-derived neural cells present an opportunity to study the role of GPR110 activation in human neurodevelopment and neuro-dysfunction. During this period, we also examined the GPR110 expression in human cortex and hippocampal tissue samples of different ages obtained from a brain bank. According to TaqMan qPCR, the GPR110 mRNA is expressed in human cortex and hippocampus, and the level is especially elevated in samples with serious brain injury, suggesting the injury-response nature of GPR110 in human brain that was observed in mouse brains after injury. We began a clinical trial in collaboration with USUHS to investigate the effect of docosahexaenoic acid supplementation on the plasma DHA metabolite level and the pregnancy outcome in women at high risk for complications due to high BMI (> 30 kg/m2) or history of preterm delivery. Tentative results indicated a strong correlation between free DHA and synaptamide, an anti-inflammatory metabolite of DHA, in plasma of these subjects.