human fibroblast cell line (normal cell line and ps1-overexpressing cell line)  (Coriell Institute for Medical Research)

Journal: Nature Communications

Article Title: Neuronal SphK1 acetylates COX2 and contributes to pathogenesis in a model of Alzheimer’s Disease

doi: 10.1038/s41467-018-03674-2

Figure Lengend Snippet: 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.

Article Snippet: PS1 and ApoE4-iPSCs were established from the patient’s skin fibroblasts (Coriell Institute) as previously described .

Techniques: Activity Assay, Isolation, Immunofluorescence, Western Blot

Journal: Nature Communications

Article Title: Neuronal SphK1 acetylates COX2 and contributes to pathogenesis in a model of Alzheimer’s Disease

doi: 10.1038/s41467-018-03674-2

Figure Lengend Snippet: Elevation of SphK1 reduces Aβ deposition and neuroinflammation in APP/PS1 mice. a Left, representative immunofluorescence images of thioflavin S (ThioS, Aβ plaques) in cortex and hippocampus of APP/PS1 and APP/PS1/SphK1 tg mice. Scale bars, 200 μm. Right, quantification of area occupied by Aβ plaques ( n = 7–8 per group). b Representative immunofluorescence images and quantification of 6E10 ( n = 5–6 per group; Scale bars, 80 μm). c Left, immunofluorescence images of microglia (Iba1) in cortex of WT, APP/PS1, APP/PS1/SphK1 tg or SphK1 tg mice brain. Low-magnification scale bars, 100 μm; High-magnification scale bars, 30 μm. Right, quantification of microglia ( n = 8–10 per group). d Left, immunofluorescence images of astrocyte (GFAP) in cortex of mice brain. Scale bars, 100 μm. Right, quantification of astrocytes ( n = 7–8 per group). e mRNA levels of inflammatory markers in cortex of mice brain ( n = 4–8 per group). Pro-inflammatory marker: TNF-α, IL-1β, IL-6 and iNOS , Immunoregulatory cytokine: IL-10 , Anti-inflammatory marker: IL-4, TGF-β and Arg1 . All data analysis was performed on 9-month-old mice. a , b Student’s t test. c – e 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.

Article Snippet: PS1 and ApoE4-iPSCs were established from the patient’s skin fibroblasts (Coriell Institute) as previously described .

Techniques: Immunofluorescence, Marker

Journal: Nature Communications

Article Title: Neuronal SphK1 acetylates COX2 and contributes to pathogenesis in a model of Alzheimer’s Disease

doi: 10.1038/s41467-018-03674-2

Figure Lengend Snippet: 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. [ 14 C] aspirin-treated neuron was positive control. Sonicated neurons incubated in the presence of [ 14 C] 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.

Article Snippet: PS1 and ApoE4-iPSCs were established from the patient’s skin fibroblasts (Coriell Institute) as previously described .

Techniques: Enzyme-linked Immunosorbent Assay, Derivative Assay, Acetylation Assay, Positive Control, Sonication, Incubation, Purification, Tandem Mass Spectroscopy

Journal: Nature Communications

Article Title: Neuronal SphK1 acetylates COX2 and contributes to pathogenesis in a model of Alzheimer’s Disease

doi: 10.1038/s41467-018-03674-2

Figure Lengend Snippet: Increased SphK1 improves microglial phagocytosis. a Colocalization of microglia (Iba1, red) with Aβ (Aβ42, 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 (Aβ 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 Aβ-loaded phagolysosomes and Aβ encapsulated in phagolysosomes ( n = 5 per group). d Morphology of microglia (Iba1, red) surrounding Aβ (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 Aβ. Down, Imaris-based automated quantification of microglial morphology ( n = 7–8 per group). e Morphometric analysis of Aβ 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.

Article Snippet: PS1 and ApoE4-iPSCs were established from the patient’s skin fibroblasts (Coriell Institute) as previously described .

Techniques: Incubation, Immunofluorescence, Quantitation Assay, Labeling