Fig 2: N-AS acetylates S565 in COX2.a N-AS binding activity of COX2 WT, COX2 S565A, COX2 N181A, COX2 T564A, and COX2 S567A was analyzed by filter binding assay. The binding velocity (Vbinding) of [14C] N-AS to COX2 was plotted to the N-AS concentration and the nonlinear regression analysis of the saturated plot yielded the kinetic parameters such as Kcat (catalytic constant) and KM (Michaelis–Menten constant) for N-AS and COX2 binding activity (n = 3 independent experiments per group). b Acetylation assay of purified COX2 protein treated with [14C] N-AS. The purified COX2 protein incubated in the presence of [14C] N-AS for 2 h at 37 °C and then COX2 was analyzed on scintillation counter. [14C] aspirin-treated COX2 protein and [14C] acetyl-CoA treated COX2 protein with SphK1 and sphingosine were positive control (n = 6–9 per group). c LC-MS spectra of peptide 560-GCPFTSFSVPDPELIK-575 (m/z = 918.94) of COX2 acetylated by N-AS. d LC-MS/MS spectra of ac-S565 in 560-GCPFTSFSVPDPELIK-575 of COX2. e Recombinant COX2 protein was incubated in the presence or absence of 2 mM N-AS or aspirin as indicated and S565 acetylation was determined by western blotting using anti-ac-S565 and COX2 antibodies. Data were replicated in six independent experiments with similar results. f The theoretical crystallographic model of human COX2 structure was taken from Protein Data Bank file 1V0X. S565 and residues (N181, T564, and S567) involved in the catalysis of COX2 were projected on the crystal structure of COX2 using PyMOL. Residues are shown as well: (1) S565 (red); (2) N181, T564, and S567 (green). g Proposed mechanism for the acetylation of S565 of COX2 by N-AS. h Acetylation assay of COX2 WT, COX2 S565A, COX2 N181A, COX2 T564A, and COX2 S567A treated with [14C] N-AS. (n = 6 independent experiments per group). b, h One-way analysis of variance, Tukey’s post hoc test. All error bars indicate s.e.m. Source data are provided as a Source data file.

Fig 3: Neuronal SphK1 induces SPMs secretion by COX2 acetylation. a Protein levels of LxA4, RvE1, and RvD1 were detected by using ELISA in CM of neurons derived from WT, APP/PS1, APP/PS1/SphK1 tg, and SphK1 tg mice (n = 6–8 per group). b mRNA levels of COX2 and LOX-15 in neurons derived from cortex of WT, APP/PS1, APP/PS1/SphK1 tg, and SphK1 tg mice (n = 4–6 per group). c Quantification of neuronal COX2 (n = 6 per group) and neuronal LOX-15 (n = 4–6 per group). d Acetylation assay of COX2 protein in neurons derived from WT, APP/PS1, APP/PS1/SphK1 tg, and SphK1 tg mice. [14C] aspirin-treated neuron was positive control. Sonicated neurons incubated in the presence of [14C] acetyl-CoA for 2 h at 37 °C and then COX2 was purified and analyzed on scintillation counter (n = 3–6 per group). e Representative chromatograms of blank, 15-R-LxA4 standard, and 15-R-LxA4 in WT samples (left panel). Molecular MS scanning from the peak at retention time 6.8 min (right upper panel) and MS/MS fragmentation pattern of 15-R-LxA4 from the peak at retention time 6.8 min (right lower panel). f Representative chromatograms (left panel) and quantification of 15-R-LxA4 in neurons derived from WT, APP/PS1, APP/PS1/SphK1 tg, and SphK1 tg mice with acetyl-coA treatment (24 h after 2.5 mM acetyl-CoA treatment) (right panel, n = 6 per group). All data analysis was done on 9-month-old mice. a-d, f One-way analysis of variance, Tukey’s post hoc test. *P < 0.05, ***P < 0.001. All error bars indicate s.e.m.

Fig 4: Increased SphK1 improves microglial phagocytosis. a Colocalization of microglia (Iba1, red) with Aß (Aß42, green) and quantification. Scale bars, 10 µm; 3D reconstruction from confocal image stacks scale bars, 10 µm (n = 7 per group). b Left, representative photomicrograph of live slice section incubated with fluorescent beads (green). Scale bar, 10 µm. White arrow point to phagocytotic microglia with fluorescent beads. Right, quantification of the number of microglial phagocytes normalized to the total number of microglia (n = 4–6 per group). c Up, immunofluorescence images of thio S (Aß plaques, green) encapsulated within Lamp1+ structures (phagolysosomes, blue) in microglia (Iba1, red) present in brains of APP/PS1 and APP/PS1/SphK1 tg mice. Low-magnification scale bars, 50 µm; High-magnification scale bars, 10 µm; 3D reconstruction from confocal image stacks scale bars, 10 µm. Down, quantitation of microglial volume occupied by Lamp1+ phagolysosomes, percent of microglia containing Aß-loaded phagolysosomes and Aß encapsulated in phagolysosomes (n = 5 per group). d Morphology of microglia (Iba1, red) surrounding Aß (ThioS, green) in cortex of APP/PS1 and APP/PS1/SphK1 tg mice. Up, high magnification (Scale bars, 10 µm) and Imaris-based three-dimensional images (Scale bars, 5 µm) of microglia surrounding Aß. Down, Imaris-based automated quantification of microglial morphology (n = 7–8 per group). e Morphometric analysis of Aß plaques in APP/PS1 and APP/PS1/SphK1 tg mice (n = 5–6 per group). Brain sections were labeled with thio S and plaques were counted and assigned to three mutually exclusive size categories based on maximum diameter: small <25 µm; medium 25–50 µm; or large >50 µm. All data analysis was performed on 9-month-old mice. a, c-e, Student’s t test. b One-way analysis of variance, Tukey’s post hoc test. *P < 0.05, **P < 0.01, ***P < 0.001. All error bars indicate s.e.m.

Fig 5: SphK1 is decreased in APP/PS1 mice neuron. a SphK1 (n = 6–7 per group) and SphK2 mRNA (n = 5–7 per group) and SphK activity (n = 6–7 per group) in cortex of brain. b Detection of sphingosine (n = 6–7 per group) and S1P (n = 6–7 per group) in brain. c SphK1 and SphK2 mRNA (n = 4 per group) and SphK activity (n = 4–10 per group) in neurons, microglia, and astrocytes isolated from mouse brain. d Detection of sphingosine (n = 4–8 per group) and S1P (n = 4–8 per group) in neurons, microglia, and astrocytes isolated from mouse brain. e Detection of SphK activity, sphingosine and S1P in neurons isolated from WT, SphK1-/-, SphK2-/- and SphK1 tg mouse brain (n = 4 per group). f Left, representative immunofluorescence images of cortex showing SphK1 (green) merged with neuron (NeuN, red). Scale bars, 20 µm. Right, quantification of neuronal SphK1 (n = 5 per group). g Western blotting for SphK1 in neuron isolated from mouse brain (n = 4 per group). All data analysis was performed on 9-month-old mice. a–g One-way analysis of variance, Tukey’s post hoc test. *P < 0.05, **P < 0.01, ***P < 0.001. All error bars indicate s.e.m.