We have previously demonstrated that DHA metabolism to N-docosahexaenoylethanolamine (synaptamide) is a significant endogenous mechanism for promoting neurogenesis, neuritogenesis and synaptogensis in a cAMP dependent manner. We demonstrated that orphan G-protein coupled receptor 110 (GPR110, ADGRF1) is the synaptamide receptor. Synaptamide specifically triggered cAMP production with low nM potency. During this period, we generated GPR110 KO mice, and demonstrated that synaptamide-induced bioactivity is abolished, confirming GPR110 as the synaptamide receptor. In addition, we demonstrated that GPR110 KO mice exhibit memory deficit evaluated by novel object recognition and Morris water maze tests. GPR110 deorphanized as a functional synaptamide receptor provides a novel target for neurodevelopmental control and offers new insight into mechanisms by which omega-3 fatty acids promote brain development and function. Growing evidence also suggests that metabolites derived from docosahexaenoic acid (DHA) have anti-inflammatory and pro-resolving effects, however, the possible role of synaptamide in inflammation is largely unknown. During this period, we tested this possibility using a lipopolysaccharide (LPS)-induced neuroinflammation model both in vitro and in vivo. For in vitro studies, we used P3 primary rat microglia and immortalized murine microglial cells (BV2) to assess synaptamide effects on LPS-induced cytokine/chemokine/iNOS (inducible nitric oxide synthase) expression by quantitative PCR (qPCR) and enzyme-linked immunosorbent assay (ELISA). To evaluate in vivo effects, mice were intraperitoneally injected with LPS followed by synaptamide, and expression of proinflammatory mediators was measured by qPCR and western blot analysis. Activation of microglia and astrocyte in the brain was examined by Iba-1 and GFAP immunostaining. We found that synaptamide significantly reduces LPS-induced production of TNF-alpha and NO in cultured microglia cells. Synaptamide increased intracellular cAMP levels, phosphorylation of PKA, and phosphorylation of CREB, but suppressed LPS-induced nuclear translocation of NF-kappaB p65. Conversely, adenylyl cyclase or PKA inhibitors abolished the synaptamide effect on p65 translocation as well as TNF-alpha and iNOS expression. Administration of synaptamide following LPS injection (i.p.) significantly reduced neuroinflammatory responses, such as microglia activation and mRNA expression of inflammatory cytokines, chemokine and iNOS in the brain. In conclusion, our study indicated that DHA-derived synaptamide is a potent suppressor of neuroinflammation in an LPS-induced model, by enhancing cAMP/PKA signaling and inhibiting NF-kappaB activation. The anti-inflammatory capability of synaptamide may provide a new therapeutic avenue to ameliorate the inflammation-associated neurodegenerative conditions. During this period, we demonstrate that ethanol at pharmacologically relevant concentrations down regulates cAMP signaling in neural stem cells (NSC) and impairs neurogenic differentiation. In contrast, synaptamide reverses ethanol-impaired NSC neurogenic differentiation through counter-acting on the cAMP production system. NSC exposure to ethanol (25-50 mM) for 4 days dose-dependently decreased the number of Tuj-1 positive neurons and PKA/CREB phosphorylation with a concomitant reduction of cellular cAMP. Ethanol-induced cAMP reduction was accompanied by the inhibition of G-protein activation and expression of adenylyl cyclase (AC) 7 and AC8, as well as PDE4 upregulation. In contrast to ethanol, synaptamide increased cAMP production, GTPgammaS binding, and expression of AC7 and AC8 isoforms in a cAMP-dependent manner, offsetting the ethanol-induced impairment in neurogenic differentiation. These results indicate that synaptamide can reduce ethanol-induced impairment of neuronal differentiation by counter-affecting shared targets in G-protein coupled receptor (GPCR)/cAMP signaling. The synaptamide-mediated mechanism observed in this study may offer a possible avenue for ameliorating the adverse impact of fetal alcohol exposure on neurodevelopment. During this period, we also established a method to probe the structure of GPR110 in living cells by combining quantitative mass spectrometry with in-cell chemical cross-linking. HEK cells expressing human GPR110 tagged with HA were subsequently treated with synaptamide (or oleoylethanolamine control) and disuccinimidyl suberate (DSS), a lysine specific crosslinker with spacer arm length of 24 . The DSS-modified GPR110 was immunopurified using anti-HA antibody and HA peptide and subjected to SDS-PAGE/tryptic digestion and quantitative mass spectrometric analysis. A total of 23 intramolecular cross-linked lysine pairs were identified. Among them, 13 pairs from the N-terminal region, and one crosslinking the intracellular loop 3 to C-terminal region, were through-space cross-linked lysine pairs. The data indicate that the alpha carbon distance of each of these cross-linked lysine pairs is within 24 . Of note, the cross-linking of K398-K438 within the GAIN domain of the N-terminal and inter-domain cross-linking of K783-K852 increases significantly after synaptamide stimulation, indicating that the ligand binding results in conformational changes in GPR110 molecules. Specifically, an intra-subdomain structure involving K398 and K438 in the GAIN domain, and an inter-domain configuration involving K783 of IL3 and K852 of C-termini become more folded upon ligand binding to the receptor. Our results represent the first experimental data for the three-dimensional structure of GPR110 in living cells at different stages. Our previous studies have shown that synaptic plasma membrane (SPM) proteins supporting synaptic integrity and neurotransmission were down-regulated in DHA-deprived brains, suggesting an important role of DHA in synaptic function. During this report period, we demonstrated aging-induced synaptic proteome changes and DHA-dependent mitigation of such changes using mass spectrometry-based protein quantitation combined with western blot or mRNA analysis. We found significant reduction of 15 SPM proteins in aging brains including fodrin-alpha, synaptopodin, PSD-95, SV2B, SNAP25, SNAP-?, NR2B, AMPA2, AP2, VGluT1, munc18-1, dynamin-1, VAMP2, rab3A, and EAAT1, most of which are involved in synaptic transmission. Notably, the first nine proteins were further reduced when brain DHA was depleted by diet, indicating that DHA plays an important role in sustaining these synaptic proteins down-regulated during aging. Reduction of at least two of these proteins was reversed by raising the brain DHA level by supplementing aged animals with omega-3 fatty acid sufficient diet for 2 months. Our results suggest a potential role of DHA in alleviating aging-associated cognitive decline by offsetting the loss of neurotransmission-regulating synaptic proteins involved in synaptic function.
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