Sphingosine Kinase 2 Potentiates Amyloid Deposition but Protects against Hippocampal Volume Loss and Demyelination in a Mouse Model of Alzheimer's Disease

Abstract

Sphingosine 1-phosphate (S1P) is a potent vasculoprotective and neuroprotective signaling lipid, synthesized primarily by sphingosine kinase 2 (SK2) in the brain. We have reported pronounced loss of S1P and SK2 activity early in Alzheimer's disease (AD) pathogenesis, and an inverse correlation between hippocampal S1P levels and age in females, leading us to speculate that loss of S1P is a sensitizing influence for AD. Paradoxically, SK2 was reported to mediate amyloid β (Aβ) formation from amyloid precursor protein (APP) in vitro To determine whether loss of S1P sensitizes to Aβ-mediated neurodegeneration, we investigated whether SK2 deficiency worsens pathology and memory in male J20 (PDGFB-APP_SwInd) mice. SK2 deficiency greatly reduced Aβ content in J20 mice, associated with significant improvements in epileptiform activity and cross-frequency coupling measured by hippocampal electroencephalography. However, several key measures of APP_SwInd-dependent neurodegeneration were enhanced on the SK2-null background, despite reduced Aβ burden. These included hippocampal volume loss, oligodendrocyte attrition and myelin loss, and impaired performance in Y-maze and social novelty memory tests. Inhibition of the endosomal cholesterol exporter NPC1 greatly reduced sphingosine phosphorylation in glial cells, linking loss of SK2 activity and S1P in AD to perturbed endosomal lipid metabolism. Our findings establish SK2 as an important endogenous regulator of both APP processing to Aβ, and oligodendrocyte survival, in vivo These results urge greater consideration of the roles played by oligodendrocyte dysfunction and altered membrane lipid metabolic flux as drivers of neurodegeneration in AD.SIGNIFICANCE STATEMENT Genetic, neuropathological, and functional studies implicate both Aβ and altered lipid metabolism and/or signaling as key pathogenic drivers of Alzheimer's disease. In this study, we first demonstrate that the enzyme SK2, which generates the signaling lipid S1P, is required for Aβ formation from APP in vivo Second, we establish a new role for SK2 in the protection of oligodendrocytes and myelin. Loss of SK2 sensitizes to Aβ-mediated neurodegeneration by attenuating oligodendrocyte survival and promoting hippocampal atrophy, despite reduced Aβ burden. Our findings support a model in which Aβ-independent sensitizing influences such as loss of neuroprotective S1P are more important drivers of neurodegeneration than gross Aβ concentration or plaque density.

Keywords: Alzheimer's disease; myelin; neuroprotection; oligodendrocyte; sphingosine 1-phosphate; sphingosine kinase.

Figure 1.

Loss of SK2 greatly reduces Aβ production. A, Immunofluorescence colabeling with 6E10 antibody (red), thioflavin S (ThioS; green), and DAPI (blue) in hippocampus of J20 and J20.SK2Δ mice at 8 and 13 months of age. Box (closed border) shows an enlarged view of an Aβ plaque (dashed box). Scale bar, 200 μm. B, Aβ plaque burden (percentage hippocampal area covered by plaques) and (C) Aβ plaque number in 13-month-old mice. D, E, Aβ40 and (F, G) Aβ42 levels in hippocampus of 13-month-old J20.SK2Δ or J20 mice, as determined by ELISA. Aβ levels were below the limit of detection in WT or SK2Δ mice. H, Western blot for full-length APP in J20 and J20.SK2Δ mice. Statistical significance in B–H was determined by two-tailed, unpaired t tests (6 mice per group).

Figure 2.

Deletion of SK2 abates hypersynchronous activity and cross-frequency coupling deficits in J20 mice. A, Representative traces and (B) quantification of spontaneous hypersynchronous events (spikes/min), in hippocampal EEG recordings from 13-month-old mice. Arrows indicate spontaneous hypersynchronous events. Means ± SEM shown; n = 3–5 mice per group. C, Spectral power density plot of hippocampal EEG recordings over the frequency range 0–100 Hz. The signal at 60 Hz was eliminated by a power line filter. D, E, Spectral power density of θ oscillation (4–12 Hz) and (F, G) low γ oscillation (25–50 Hz) in WT, SK2Δ, J20, and J20.SK2Δ mice. Mean (solid line) and SEM (dotted line) shown in D and F; AUC shown in E and G. H, Representative co-modulograms (30 s) of EEG recordings. Color contour indicates strength of co-modulation of amplitude by phase at individual frequency pairs. I, Modulation index of cross frequency coupling between θ phase and γ frequencies in EEG recordings. Mean ± SEM. Statistical significance in B, E, G, and (I) was determined by ANOVA with Sidak's post-test. P values < 0.05 are indicated on graphs. Data were derived from three to five mice per group. J, Phase–amplitude co-modulation across θ phase bins. Mean (solid line) and SEM shown; n = 3–5. Statistical significance was determined by two-way ANOVA with Dunnett's post-test. *p < 0.05, J20 compared with WT; #p < 0.05, J20.SK2Δ compared with WT.

Figure 3.

J20.SK2Δ mice exhibit deficits in recognition memory. A, Entries into, and (B) distance traveled in, the novel arm of the Y-maze as a percentage of total arm entries and total distance traveled. C, D, Time spent nosing the inhabited cage, expressed as a percentage of total time nosing (inhabited + uninhabited), in the SPT. E, F, Time spent nosing the cage with the novel mouse, expressed as a percentage of total time nosing (novel + familiar), in the SNT. G, H, Time spent nosing the novel object, expressed as a percentage of total time nosing (novel + familiar), in the NORT. C, E, G, Seven-month-old mice; A, B, D, F, H, 12-month-old mice. Statistical significance in all paradigms was determined by one-sample t tests comparing the measure-of-interest to that expected by chance (dotted line) for each genotype (n = 10–14 mice per group). *p < 0.05, **p < 0.01, ***p < 0.001; ns, not significant.

Figure 4.

Loss of hippocampal volume in J20.SK2Δ mice. A–D) Estimated volume of the DG (A, C) and CA1–3 region (B, D) of the hippocampus in 13-month-old (A, B) and 8-month-old (C, D) mice. mth, Month. E, NeuN staining of a WT mouse hippocampus with CA and DG regions outlined. F, Mean NeuN-positive area of the hippocampus in 13-month-old mice. G, NeuN-negative area as a percentage of total hippocampal area in 13-month-old mice. Statistical significance was determined by one-way ANOVA with Sidak's post-test (5–6 mice per group). ANOVA results reported in-text with significant post-test results shown on the graphs.

Figure 5.

Pronounced hypomyelination in J20.SK2Δ mice. A–L, Immunofluorescence for MBP (green) and NF-H (red) in the cortex (A–D), hippocampus (E–H), and cerebellum (I–L) of 13-month-old WT (A, E, I), SK2Δ (B, F, J), J20 (C, G, K), and J20.SK2Δ mice (D, H, L). M–P, MBP immunofluorescence in the hippocampus of 7-month-old WT (M), SK2Δ (N), J20 (O), or J20.SK2Δ mice (P). Scale bar, 200 μm.

Figure 6.

Loss of myelin markers and reduced oligodendrocyte density in J20.SK2Δ mice. Western blots (A, C) and densitometric quantification (B, D) of myelin and neuronal markers in cortex and hippocampus of 8-month-old (A, B) and 13-month-old (C, D) mice. Protein levels were normalized to β-actin in each sample, then to the mean of the control group. Graphs show mean ± SEM; n = 5–6 mice per genotype. Statistical significance was determined by one-way ANOVA with Sidak's post-test. E, Olig2-positive cell density in the hippocampus (mean ± SEM; n = 5–6). Statistical significance was determined by two-way ANOVA with Dunnett's post-test. P values for comparisons that were significant in post-tests (p < 0.05) are shown on the graphs.

Figure 7.

Inhibition of endosomal cholesterol export affects sphingosine phosphorylation. A, Levels of S1P and sphingosine in the cortex of 13-month-old WT, SK2Δ, J20, and J20.SK2Δ mice (5–6 mice per group). Statistical significance was determined by one-way ANOVA followed by Sidak's post-test, **p < 0.01. ANOVA results for S1P: F = 9.7; p = 0.0005; sphingosine: F = 17.7, p < 0.0001. B, SK2 activity in hippocampus of 13-month-old WT or J20 mice (6 mice per group). Activity is normalized to the mean of the WT group. C, S1P and sphingosine, and (D) SK2 activity, in MO3.13 cells treated with 3 μg/ml U18666A. Levels are normalized to the mean of the zero time point. E, S1P/sphingosine ratio in U251 cells, primary astrocytes, Oli-neu cells, or SH-SY5Y cells treated with 3 μg/ml U18666A. F, G, S1P and sphingosine in MO3.13 cells treated with (F) 5 mm NH₄Cl or (G) 100 μm leupeptin. Statistical significance in B–G was determined by one-way ANOVA and reported in the results text. Each experiment involved four independent cell treatments. H, Western blots showing LC3B-I (top band) and LC3B-II (bottom band) protein levels, with β-actin as a loading control, in MO3.13 cells treated with U18666A, leupeptin, or NH₄Cl.