Alterations In Lipid Metabolism In The Nervous System By Ethanol

National Institute On Alcohol Abuse And Alcoholism

Investigators

Linked publications, trials & patents

Abstract

We have previously established that the metabolism of docosahexaenoic acid (DHA) to N-docosahexaenoylethanolamine (synaptamide) is a significant endogenous mechanism for promoting neurogenesis, neuritogenesis, synaptogenesis and anti-inflammatory action. We further demonstrated that orphan G-protein coupled receptor 110 (GPR110, ADGRF1), an adhesion GPCR (aGPCR), is the synaptamide target receptor, triggering cAMP production with low nM potency. We also established in vivo significance of this mechanism in injury/inflammation models, suggesting a therapeutic potential of targeting GPR110. During this review period, we continued to characterize the structure and function of GPR110 with particular attention to the modulation by ethanol and omega-3 fatty acid (PUFA) diet. We found that GPR110 signaling along with its developmental function is recapitulated in neural cells derived from human induced pluripotent stem cells (iPSCs), presenting an opportunity to study the role of GPR110 activation in human neurodevelopment and neuro-dysfunction. GPR110 is expressed in iPSCs, iPSC-derived neural progenitor cells (NPCs) and NPC-derived neurons, and GPR110 ligands dose-dependently increased cAMP in these cells. We generated a functionally inactive GPR110 mutant iPSC line with a mutation at F663S presumably associated with schizophrenia based on the Swedish schizophrenia exome-sequencing study. During this report period, we continued to investigate the relevance of GPR110 signaling in human neurodevelopment using WT and F663S mutant human NPCs along with GPR110 KO mouse model. We found that ligand-induced GPR110 activation stimulated neurogenesis and neurite growth in WT but not in F663S mutant NPCs. GPR110 KO mice in vivo or neurons derived from inactive F663S mutant NPCs showed abnormal developmental phenotypes according to RNA sequencing, imaging analysis and western blot analysis. In addition to the downregulated neurogenesis and neurite growth, the expression of glutamate receptors and synaptic molecules associated with the postsynaptic density were significantly reduced in GPR110 KO mouse brain or neurons derived from human NPC with the F663S mutation. In contrast, the F663S mutant neurons showed increased expression of genes implicated in neuropsychiatric disorders. Microelectrode array (MEA) assay that monitors spontaneous electrophysiological activity of neurons also revealed decreased synaptic activity in the F663S mutant compared to the WT neurons. Our results indicate a significant role of GPR110 and its downstream signaling in neuronal differentiation and neuromaturation during development. The lack of GPR110 signaling leading to aberrant neuronal development may contribute to the psychiatric abnormality in the adult stage. We have previously demonstrated that ethanol exposure leads to impaired neurogenesis in mouse neural stem cells (NSCs) and synaptamide reverses the adverse impact of ethanol. During this report period, we extended the investigation to human NPCs. As was the case with mouse NSCs, human NPCs exposed to 25-50 mM EtOH for 3 days (DIV1-3) significantly reduced neurogenesis, and the neurons derived from the ethanol-exposed NPCs exhibited significantly less neurite ramification compared to the control neurons. We also found that neurons exposed to ethanol in early stage (DIV3-5) showed significantly decreased neuronal activity at a later stage (DIV20), evaluated by the MEA assay. These results indicate that pharmacologically relevant concentrations of EtOH impair neurogenesis and neuromaturation even if human NPCs were exposed briefly at an early stage. GPR110 activation by synaptamide may revert the adverse impact of ethanol on neuronal development of human NPCs as observed for mouse NSCs. Although the significance of GPR110 in neurodevelopment and neuroprotection has begun to unfold, most biological functions of this receptor are still unknown. Recently, we have discovered significant involvement of GPR110 in blood-brain barrier (BBB) function. As GPR110 is highly expressed also in the kidney, we investigated whether GPR110 similarly affects kidney function during this report period. We found that the albumin to creatinine ratio observed in urine samples from GPR110 KO mice was markedly elevated, indicating dysfunction of the glomerular filtration barrier. Moreover, the injection of lipopolysaccharide (LPS, 1 mg/kg, i.p.) exacerbated albumin leakage in urine, presumably due to inflammatory responses caused by LPS. To unveil the molecular basis of this renal phenotype presented by GPR110 KO, we conducted a comparative analysis of kidney protein expression between GPR110 KO and WT mice through label-free quantitative LC-MS/MS, followed by pathway analysis. Over 13,000 proteins were identified, and 300 proteins showed significant differences in their level (p<0.05, fold change 1.25, n = 8). Pathway analysis revealed 16 entities associated with increased albumin permeation or glomerular injury. Notably, vascular endothelial growth factor A (VEGFA) was found to be increased by nearly twofold in GPR110 KO mice, which was confirmed by western blotting. We also observed the elevated phosphorylation of VEGF receptor 2 (VEGFR2) in the GPR110 KO kidney. It is well established that VEGFA, that is highly expressed in glomerular podocytes and tubular epithelial cells, triggers VEGFR2 signaling, subsequently enhancing vascular permeability in the kidney. Notably, LPS treatment further amplified VEGFA expression, VEFGR2 phosphorylation and its downstream signaling such as phosphorylation of Src and p38-MAPK. In summary, we identified glomerular dysfunction indicated by proteinuria as a renal phenotype associated with GPR110 KO. Depletion of GPR110 elevated VEGFA levels that in turn upregulated VEGFR2 signaling, a known mediator of increased vascular permeability leading to albuminuria. The regulation of VEGFA-VEGFR2 signaling may be an important physiologic mechanism by which GPR110 controls glomerular filtration barrier function. Previously, we have found exacerbating effects of single binge ethanol exposure on LPS-induced neuroinflammation which can be ameliorated by synaptamide in a GPR110-dependent manner by counteracting the effect of ethanol and LPS on the NLRP3 expression and cAMP system. During this review period, we extended our research to investigate whether dietary manipulation of brain DHA status influences the exacerbating effect of acute single binge ethanol exposure on LPS-induced neuroinflammation. To manipulate the brain DHA status, pregnant dams of GPR110 WT and KO mice were placed on an omega-3 adequate or deficient diet from gestational day 13 throughout the pregnancy and lactation period and offspring mice were weaned on the respective diets. At 10 to 12 weeks of age, mice were administered either ethanol (3 g/kg, gavage) or maltose (5.4 mg/kg, gavage). After 4 h, mice were injected with LPS (1 mg/kg, i.p.) and brains were collected 2 h later. Expression of inflammatory markers (TNF- and IL-1) as well as PDE4b after single binge ethanol administration followed by LPS injection was significantly higher in the brains from WT animals fed the deficient diet compared to the adequate diet group. However, in KO animals such diet effects were not observed. The levels of DHA and synaptamide in the brain were significantly lower in mice on the omega-3 deficient diet, but there was no noticeable difference in lipid profiles between GPR110 KO and WT mice. These data indicated a beneficial effect of GPR110 activation by synaptamide endogenously derived from DHA on LPS-induced neuroinflammation enhanced by single binge ethanol exposure. A diet rich in omega-3 PUFA may serve as a preventive strategy to ameliorate neuroinflammation caused by LPS and ethanol.