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 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 GPCRs (aGPCRs), is the target receptor mediating bioactivity of synaptamide by triggering cAMP production with low nM potency. We established in vivo significance of this mechanism in neurodevelopment enhancing cognitive function. GPR110 activation also led to suppression of neuroinflammation after brain injury, suggesting a therapeutic potential of targeting GPR110. We confirmed 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. We generated a GPR110 mutant iPSC line with F663S mutation that was detected in schizophrenia case from the Swedish schizophrenia exome-sequencing study. We found that ligand-induced GPR110 activation stimulated neurogenesis and neurite growth in WT but not in F663S mutant NPCs. The F663S mutant neurons exhibited abnormal developmental phenotypes with decreased synaptic activity and increased expression of genes implicated in neuropsychiatric disorders. We have also discovered impaired blood-brain barrier (BBB) function in GPR110 knockout mice (KO). The lack of GPR110 signaling leading to aberrant neuronal development as well as compromised BBB function may contribute to the psychiatric abnormality in the adult stage. We also established an essential function of GPR110 in kidney glomeruli where its expression is high throughout the life span. Proteinuria was identified as a renal phenotype associated with GPR110 KO. Depletion of GPR110 elevated proinflammatory signals and VEGFA expression that in turn upregulated VEGFR2 signaling, a known mediator of increased vascular permeability leading to albuminuria. The regulation of VEGFA-VEGFR2 signaling by GPR110 is an important physiological mechanism to control glomerular filtration barrier function. 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 polyunsaturated fatty acid (PUFA) diet. GPR110, an adhesion G protein-coupled receptor (aGPCR), contains a self-cleaving SEA (Sperm protein, Enterokinase, and Agrin) domain (aa 148–256) positioned upstream of the conserved GPCR-autoproteolysis-inducing (GAIN) domain (aa 256–578) in its extracellular region. While GAIN domain autoproteolysis is important for aGPCR activation, the role of SEA domain cleavage and its biological significance remain poorly understood. Using mutagenesis and quantitative mass spectrometry, we identified that the SEA domain undergoes self-cleavage between residues G206 and S207. Notably, substitution of S207 with alanine (S207A) not only abolished SEA domain cleavage but also reduced GAIN domain autoproteolysis. Similarly, deletion of the C-terminal fragment of the SEA domain (Δ207–256) or removal of the entire SEA domain produced the same effect. These results indicate that both the presence of the SEA domain and its cleavage are critical for GAIN domain autoproteolysis. It is possible that the C-terminal fragment of the SEA domain remains noncovalently associated and participates in GAIN domain autoproteolysis. Alternatively, the exposure of the N-terminal side of the cleavage site in SEA domain (148–206) may be required for the autoproteolytic activity of the GAIN domain. These mutations also affected downstream signaling. Bioluminescence resonance energy transfer (BRET)-based assays revealed that the mutations impaired basal GPR110/Gq signaling activity, which depends on GAIN domain autoproteolysis, while Gs signaling induced by synaptamide-like ligands remained unaffected. These findings reveal the biological importance of the GPR110 SEA domain in regulating GAIN domain autoproteolysis and Gq-mediated downstream signaling. During this period, we also continued to investigate the effects of GPR110 signaling on mouse neurobehavioral outcome. In addition to KO mice, we have generated various GPR110 knock-in (KI) mouse models with overexpressed GPR110 in specific brain regions or cell types. We demonstrated that GPR110 KO impairs learning and memory both in male and female mice, indicating that GPR110 plays a critical role in cognitive function across sexes. GPR110 KO mice also exhibited increased anxiety-like behaviors, as evidenced by greater immobility in the open field test, although no statistically significant differences were observed in the time spent in the center of the field. Conversely, overexpression of GPR110 in glutamatergic neurons achieved by crossing GPR110 KI mice with VGluT2 mice reduced anxiety-like behaviors, indicated by decreased marble burying behavior. To explore the implication of the inactive human mutation of GPR110 found in a Swedish schizophrenia case-controlled exome-sequencing study, schizophrenia-like behaviors in GPR110 KO mice were evaluated using the prepulse inhibition test during this period. Preliminary results indicated a trend towards increased startle response and decreased inhibition in GPR110 KO mice, mimicking schizophrenia-like behaviors. We also tested LPS induced anxiety-like behaviors in mice with microglial overexpression of GPR110 produced by crossing GPR110 KI with CX3CR1 mice using open field and elevated plus maze tests. After LPS injection, mice without GPR110 overexpression showed a significant decrease in the time spent in center or open arms while increasing the immobility time. With microglial overexpression of GPR110, these changes were not observed, indicating an ameliorating effect on LPS-induced anxiety-like behaviors. To understand molecular mechanisms for the observed behavioral effects of GPR110, we investigated hippocampus and amygdala proteome changes using quantitative mass spectrometry. Differential proteome analysis by comparing wild-type (WT) and GPR110 KO revealed significant alterations in 300 proteins in GPR110 KO mice relative to WT (p < 0.05, fold change ≥ 1.20; n = 4 WT, n = 3 KO) in hippocampus although proteomic changes in the amygdala were less pronounced. Of these, 200 proteins were downregulated and 100 were upregulated in the KO group. Many of the downregulated proteins in GPR110 KO mice were associated with synaptogenesis and glutamatergic receptor pathways, several of which are critical for CREB signaling. Additional changes were observed in proteins involved in neurotransmission, synaptic formation, and vesicle trafficking. Pathway analysis corroborated behavioral findings, showing significant downregulation of processes related to cognition, learning, memory, and prepulse inhibition in KO mice. Additionally, pathways involved in neurogenesis, neuronal morphogenesis, neural growth, dendritic branching, and neuritogenesis were also reduced. In contrast, mass spectrometry analysis of amygdala samples did not reveal significant differences in anxiety-related pathways. These results provide molecular insights into the role of GPR110 in the hippocampal proteome and highlight its essential contribution to brain function. Collectively, our findings suggest that GPR110 is essential for learning and memory and influences certain anxiety-like behaviors. Alterations in the hippocampal proteome, particularly those affecting neurogenesis, neuritogenesis and synaptogenesis may have contributed at least in part to the observed behavioral changes due to GPR110. Prenatal alcohol exposure is known to affect behavioral outcomes in adulthood. We have previously demonstrated that ethanol exposure leads to impaired neurogenesis in mouse neural stem cells (NSCs) and DHA-derived synaptamide reverses the adverse impact of ethanol. During this review period, we extended our research to investigate the effects of perinatal ethanol exposure and dietary omega-3 polyunsaturated fatty acids (PUFA) on neurobehavioral outcomes in mice. To this end, we established three experimental groups receiving maternal exposure to ethanol or water from gestational day 10 to postnatal day 10: (1) mice receiving a PUFA-sufficient diet both prenatally and postnatally, (2) mice maintained on a PUFA-deficient diet throughout development, and (3) mice switched from a PUFA-deficient diet to a sufficient diet at weaning. Our results indicated that a PUFA-sufficient diet can ameliorate the ethanol effects on anxiety-like behaviors and learning and memory function. Ethanol tended to decrease the time spent in the open arms of the elevated plus maze in the PUFA-deficient and deficient then sufficient diet groups but not in the PUFA-sufficient group. Similarly, alcohol decreased the discrimination index compared to water-drinking controls in the novel object recognition test among mice fed PUFA-deficient diet, but this deficit was not observed in the group maintained on a PUFA-sufficient diet. Interestingly, the effect of alcohol was more prominent in male mice. On the contrary, females tended to be more sensitive to diet effects, performing worse on the test when given the deficient diet. Abnormalities in neurodevelopment have been described as part of the pathophysiology of schizophrenia. DHA, highly enriched in the brain, is known to facilitate optimal neurodevelopment and function. Its endogenous metabolite synaptamide promotes neurogenesis and synaptogenesis and improves cognitive function through GPR110 activation. The lack of GPR110 activity was found to be associated with neurodevelopmental abnormalities, blood brain barrier impairment and schizophrenia. Besides the receptor manipulation, endogenous metabolism of DHA to its ligand synaptamide can alter GPR110 signaling and biological outcomes. Therefore, in collaboration with the Feinstein Institutes for Medical Research, we have investigated the possible link between PUFA metabolism and the pathophysiology of schizophrenia by determining the concentration of unesterified (free) PUFAs and their metabolites including arachidonic acid (AA), DHA, anandamide and synaptamide in the cerebrospinal fluid (CSF) and matching plasma samples from a group of patients with schizophrenia-spectrum disorder (SSD) diagnosis and healthy volunteers. We found that free AA levels in CSF were significantly elevated in SSD patients compared to healthy controls. Higher median levels of free DHA and synaptamide were also observed in CSF from the patient group, but these differences were not statistically significant. There was a significant correlation between CSF DHA and CSF AA with age, and between CSF levels of synaptamide and its precursor free DHA, suggesting age-dependent release of free PUFA and biosynthesis of DHA-derived synaptamide in the human brain. The levels of these metabolites in matching CSF and plasma significantly correlated, and this correlation was particularly stronger in patients compared to controls, supporting earlier findings that blood-brain barrier permeability is increased in GPR110 KO mice and schizophrenia patients. The neurodevelopmental and neurobehavioral significance of GPR110 signaling that we established in mouse and human iPSC-derived neural cell models as well as in humans may provide a mechanistic clue to neuropsychiatric disorders like schizophrenia. As a continuing effort to develop a novel therapeutic strategy based on GPR110 signaling, we investigated the effects of dimethylsynaptamide (DMS), a synthetic GPR110 ligand, in encephalomyelitis (EAE) as a model in collaboration with NCATS and the Providence Brain and Spine Institute. We found that DMS crossed the blood brain barrier (BBB), significantly reduced the severity of clinical EAE scores, and delayed clinical disease EAE onset as compared to saline and vehicle-treated controls. DMS administration also reduced pro-inflammatory cytokine expression in both periphery and CNS, lowered meningeal, perivascular and parenchymal T-cell lymphocyte aggregation and decreased microglial/macrophages and astrocyte activation in the spinal cord. Lowered clinical disability along with suppressing effects of DMS on innate and adaptive immune system in CNS revealed in our study may support the potential therapeutic application of DMS for MS. In summary, our study indicated that GPR110 signaling is an important physiologic mechanism for neurodevelopment and neuroprotection as well as kidney glomerular barrier function. Suppression of GPR110 signaling by receptor mutation or ligand depletion can lead to neurodevelopmental and neuropsychiatric abnormality while ligand-induced GPR110 activation may present a novel strategy for CNS injury or diseases associated with neuroinflammation.