Orphan GPR110 (ADGRF1) targeted by N-docosahexaenoylethanolamine in development of neurons and cognitive function

Abstract

Docosahexaenoic acid (DHA, 22:6n-3) is an omega-3 fatty acid essential for proper brain development. N-docosahexaenoylethanolamine (synaptamide), an endogenous metabolite of DHA, potently promotes neurogenesis, neuritogenesis and synaptogenesis; however, the underlying molecular mechanism is not known. Here, we demonstrate orphan G-protein coupled receptor 110 (GPR110, ADGRF1) as the synaptamide receptor, mediating synaptamide-induced bioactivity in a cAMP-dependent manner. Mass spectrometry-based proteomic characterization and cellular fluorescence tracing with chemical analogues of synaptamide reveal specific binding of GPR110 to synaptamide, which triggers cAMP production with low nM potency. Disruption of this binding or GPR110 gene knockout abolishes while GPR110 overexpression enhances synaptamide-induced bioactivity. GPR110 is highly expressed in fetal brains but rapidly decreases after birth. GPR110 knockout mice show significant deficits in object recognition and spatial memory. GPR110 deorphanized as a functional synaptamide receptor provides a novel target for neurodevelopmental control and new insight into mechanisms by which DHA promotes brain development and function.

Figure 1. cAMP-dependent induction of neurite outgrowth, synaptogenesis and neurogenesis by synaptamide.

In cortical neurons, 10 nM synaptamide (Syn) increased cAMP production, neurite outgrowth (a) and synaptogenesis (b) while pretreatment with SQ22,536 for 30 min prevented these effects. Neurite outgrowth and synaptogenesis were evaluated after treating neurons from day 1 in vitro (DIV1) for 2 or 6 days, respectively, while cAMP production and CREB phosphorylation were determined after stimulating DIV3 neurons with synaptamide for 10 min. Neurons and nuclei were visualized using MAP2 antibody (green) and DAPI (blue). Co-localization analysis of presynaptic marker synapsin1 (red) and postsynaptic marker PSD95 (green) revealed increased synapses (overlapping synapsin1/PSD95 puncta indicated by arrows) by synaptamide treated cortical neurons but not in the presence of SQ22,536 (b). For quantification of synaptic protein puncta, 20 cells were scored per well for 3 wells per each group. Synaptamide at 10 nM induced G-protein activation (c). The [γ-³⁵S] GTP binding was measured after treating NSC membranes with 10 nM synaptamide for 15 min with or without overnight pretreatment with pertussis toxin (PTX, 0.1 μg ml⁻¹). While expected increases in cAMP production were observed after PTX pretreatment of cortical cells or NSCs, synaptamide remained capable of increasing cAMP (d). Values are expressed as means±s.e.m. of biological triplicates (n=3), representing three independent experiments. Statistical analysis was performed using unpaired Student t-test. ^#P<0.05, ^##P<0.01, ^###P<0.001, *P<0.05, **P<0.01, ***P<0.001 in comparison with the corresponding control. ¶, relative to control. Scale bars, 20 μm (a) and 5 μm (b).

Figure 2. Identification of synaptamide receptor and associated G-protein.

Biotinylated synaptamide analogue G1 (a) increased cAMP production in DIV3 neurons after 10 min stimulation (b). After mouse fetal brains or NSCs were lysed in PBS containing 0.5% Triton X-100, treated with G1, affinity-purified using streptavidin beads and subjected to SDS/PAGE, the entire gel was cut into several bands for tryptic digestion and protein identification (ID) by mass spectrometric analysis (c). The MS/MS spectra of GPR110 peptides G(432–442)R and G(460–471)K detected from the gel band in the 100–130 kD molecular weight region are shown (d). Binding of G1 to GPR110 was confirmed by the western blot detection of mGPR110-HA expressed in HEK cells at ∼100 kD after streptavidin pull-down while synaptamide dose-dependently decreased the mGPR110-HA recovered after G1-streptavidin pull-down (e). Among fatty acid ethanolamides, only synaptamide displaced the G1 binding to mGPR110 (f). GPR110 expression in the plasma membrane was detected (g). The cAMP response induced by 10 nM synaptamide increased in a GPR110 gene-dose-dependent manner (h). GPR110 interaction with Gαs was identified by co-immunoprecipitation of overexpressed mGPR110-HA with endogenous Gαs (i). Syn, synaptamide; n3, omega-3; n6, omega-6; DPEA, N-docosapentaenoylethanolamine; OEA, N-oleoylethanolamine; AEA, N-arachidonylethanolamine. Results in d and i represent two independent experiments. Data in (b,e,f,h) are means±s.e.m. of biological triplicates (n=3), representing three independent experiments. **P<0.01, ***P<0.001 versus control, and ^#P<0.05, ^##P<0.01, ^###P<0.001 versus G1 binding by unpaired Student t-test. ¶, relative to control. Scale bar, 5 μm (g).

Figure 3. Synaptamide binding to GPR110 in living cells identified by in-cell cross-linking/affinity purification or fluorescence microscopy.

Chemical structure of the biotinylated synaptamide analogue (G1*) containing a cross-linkable primary amine group (a). G1* and synaptamide at 10 nM similarly induced cAMP production in DIV3 cortical neurons after a 10-min incubation (b). Strategy to detect G1* bound-GPR110 in living cells (c). The G1*-receptor complex stabilized by DSS cross-linking was detected by anti-HA antibody for GPR110-HA-expressing HEK cells or peroxidase conjugated streptavidin for endogenous GPR110 in NSCs. The control was treated with biotin instead of G1* (d). Chemical structure of bodipy-synaptamide (e). Similar to synaptamide, bodipy-synaptamide at 10 nM increased cAMP production in DIV3 cortical neurons after a 10-min incubation (f). Confocal images of fluorescent endocytic receptor puncta detected after incubating DIV3 cortical neurons with bodipy-synaptamide (100 nM) for 30 min (g). Only weak non-specific signal without fluorescent puncta were observed after the pretreatment with anti-GPR110 antibody (0.4 μg ml⁻¹) or synaptamide (10 nM) for 30 min, indicating specific binding of synaptamide to GPR110 on the surface of live cortical neurons (g). Synaptamide dose-dependently increased cAMP production in cortical neurons and NSCs with EC₅₀ in the low nM range (closed circle), which was blocked by 30 min pretreatment with GPR110 antibody at 0.4 μg ml⁻¹ (open circle) (h). DSS: disuccinimidyl suberate. Data represent two (d,h), three (b,f) or six (g) independent experiments. Values in (b,f,h) are means±s.e.m. of biological triplicates (n=3). **P<0.01, ***P<0.001 versus control by unpaired Student t-test. ¶, relative to control. Scale bar, 20 μm (g).

Figure 4. Significance of GPR110 N-terminus in ligand binding and synaptamide-induced cAMP production.

Human GPR110 sequence map shows wild type full size hGPR110 (1–910, WT), GPS cleavage sequence (HL̂T) double mutant (H565A/T567A, DM), N-terminal fragment (1–566, NTF) and C-terminal fragment (567–910, CTF) (a). When the N-terminus was truncated (CTF), neither binding of synaptamide analogues G1 to hGPR110 (b) nor synaptamide-induced cAMP production (c) was observed, while the C-terminal truncated form, NTF, showed G1 binding. Double mutation at GPS (DM) to prevent the autocleavage did not alter the ligand binding ability or synaptamide-induced cAMP production. The treatment with 10 nM synaptamide did not affect hGPR110 autocleavage (d). To evaluate cAMP production, various mutant forms of hGPR110 were transiently overexpressed in HEK293 cells permanently expressing CRE-luc2P as a cAMP sensor and treated with 10 nM synaptamide for 16 h. M45: empty vector expressing cells. Data represent three independent experiments (b–d). Values in c are means±s.e.m. of biological triplicates (n=3). ***P<0.001 versus the non-synaptamide treated control by unpaired Student t-test.

Figure 5. Synaptamide-induced cAMP production and neurite outgrowth affected by GPR110 knockdown, pretreatment with anti-GPR110 antibody or GPR110 overexpression.

Cortical neurons were pre-treated with 0.4 μg ml⁻¹ N-terminal targeting anti-GPR110 antibody for 30 min on DIV1 or DIV3, and incubated with 10 nM synaptamide for 48 h or 10 min for the evaluation of neurite outgrowth or cAMP production, respectively. The GPR110 antibody pretreatment abolished synaptamide-induced increases in cAMP (a), CRE activity (b) and MAP2-immunostained neurite outgrowth (c,d). Transfection of cortical cells with GFP-expressing GPR110 shRNAs (sh1 and sh2) performed on day 0 effectively suppressed GPR110 expression on DIV3 (e). shRNA expression prevented synaptamide-induced neurite outgrowth (f,g) and cAMP production (h) while scrambled control RNA (Sc) showed no effects. For cAMP assay, transfected cortical cells were treated with 10 nM synaptamide on DIV3 for 10 min. Neurite outgrowth was evaluated for transfected (GFP-positive) cells after treatment with 10 nM synaptamide on DIV1 for 2 days. Overexpression of HA-tagged mGPR110 in A549 cells or HA-tagged hGPR110 in CRE-luc2P HEK cells for 24 h (i) significantly increased cAMP production after 10 min stimulation with 10–100 nM synaptamide compared with the M45 empty-vector expressing cells (j,k). Data represent three independent experiments. Values are shown as means±s.e.m. of biological triplicates (n=3). *P<0.05, **P<0.01, ***P<0.001, ^#P<0.05, ^##P<0.01, ^###P<0.001 by unpaired Student t-test versus the corresponding control. ¶, relative to control. Scale bars, 20 μm (d,f).

Figure 6. Expression of gpr110 transcripts in the mouse brain.

Temporal and regional expression of GPR110 mRNA evaluated by quantitative real-time PCR indicated high expression in NSCs and fetal brain (FB) as well as in hippocampus (HC), particularly the dentate gyrus (DG) area, in 4 month (4 M) old adult brains (a). The expression was normalized to the level in the P0 cortex (b). In situ hybridization also indicated the considerable expression of gpr110 in P0 brain cortex and hippocampus as wells as adult hippocampal DG area while adult cortical region showed rare expression (b). Coronal sections of newborn and adult mouse brains are shown. The boxed areas in the micrographs on the far left panels are shown at a higher magnification for CX and HC regions in the two right panels. Nuclei were counterstained with hematoxylin. Positive staining is indicated by the high expression in glomeruli of P0 and adult kidney. The P0 kidney from KO mouse was used as a negative control. CX, cerebral cortex. Data represent three (a) or five (b) independent experiments. Data in a are means±s.e.m. of biological triplicates (n=3). *P<0.05, ***P<0.001 by unpaired Student t-test versus newborn (P0) cortex.

Figure 7. Synaptamide bioactivity abolished by GPR110 KO.

No expression of gpr110 in GPR110 KO kidney and brain cortex indicated by PCR and in situ hybridization (a). The endogenous synaptamide level (mean±s.d., biological triplicates, n=3) in WT and KO P0 brains determined by mass spectrometry was comparable (b). In cortical neurons from KO mice, synaptamide up to 100 nM showed no effect on cAMP production (c) or neurite outgrowth (d). Co-localization analysis of presynaptic marker synapsin1 (red) and postsynaptic marker PSD95 (green) revealed increased synapses (overlapping puncta indicated by arrows) by synaptamide treatment in WT but not in KO cortical neurons (e). For quantification of synaptic protein puncta in primary neuron culture, 20 cells were microscopically scored per condition, and the data represent three independent experiments. In vivo synaptogenesis evaluated in cortical synaptic zone of P10 mouse brain showing severe loss of synapsin1, Homer1 and PSD95 puncta as well as synapsin1/Homer1 or synapsin1/PSD95 colocalizing puncta in the KO brain, indicating significant loss of synapses (f). KO animals exhibited significantly reduced recognition memory (g) and spatial memory compared with WT (h). Schematic representation of synaptamide signalling (i). DHA-derived synaptamide binds to GPR110 which in turn triggers Gαs activation, cAMP increase and CREB phosphorylation to promote neurite growth, synaptic protein expression and NSC neurogenic differentiation. Data represent three (a–e) and two (f) independent experiments. Values are means±s.e.m. of biological triplicates (n=3) unless specified otherwise. *P<0.05, **P<0.01, ***P<0.001, ^#P<0.05, ^##P<0.01, ^###P<0.001 versus corresponding control (c–e) or WT platform quadrant time (h) by unpaired Student t-test. For the comparison of the time in four quadrants within the WT or KO group (h), one-way ANOVA followed by Tukey post hoc test was used with the statistical significance at P<0.05. Means designated with the same letter (a or b) are not significantly different. NT, N-terminus. N.S., Not significant. ¶, relative to control. Scale bars, 100 μm (a) and 5 μm (e,f).