Sodium rutin ameliorates Alzheimer's disease-like pathology by enhancing microglial amyloid-β clearance

Abstract

The accumulation of aggregated amyloid-β (Aβ) in the brain is the first critical step in the pathogenesis of Alzheimer's disease (AD), which also includes synaptic impairment, neuroinflammation, neuronal loss, and eventual cognitive defects. Emerging evidence suggests that impairment of Aβ phagocytosis and clearance is a common phenotype in late-onset AD. Rutin (quercetin-3-rutinoside) has long been investigated as a natural flavonoid with different biological functions in some pathological circumstances. Sodium rutin (NaR), could promote Aβ clearance by increasing microglial by increasing the expression levels of phagocytosis-related receptors in microglia. Moreover, NaR promotes a metabolic switch from anaerobic glycolysis to mitochondrial OXPHOS (oxidative phosphorylation), which could provide microglia with sufficient energy (ATP) for Aβ clearance. Thus, NaR administration could attenuate neuroinflammation and enhance mitochondrial OXPHOS and microglia-mediated Aβ clearance, ameliorating synaptic plasticity impairment and eventually reversing spatial learning and memory deficits. Our findings suggest that NaR is a potential therapeutic agent for AD.

Fig. 1. Preparation and characterization of NaR.

(A) Molecular structure of rutin. (B) Preparation procedures of NaR; the final concentration of NaR stock solution was 10 mg/ml. (C) HPLC analysis of NaR (0.1 mg/ml in water; injection volume was 20 μl) and rutin [0.1 mg/ml in dimethyl sulfoxide (DMSO); injection volume was 20 μl]. mAU, milli–absorbance units. (D) TLC assay of NaR and rutin. NaR(S): Solid NaR was dissolved with double-distilled water (ddH₂O) at 0.5 mg/ml and 20 μl was injected. NaR(L): NaR stock solution was diluted with ddH₂O at 0.5 mg/ml and 20 μl was injected. Rutin was dissolved with DMSO at 0.5 mg/ml and 20 μl was injected. (E) ¹³C and ¹H shift changes of NaR compared with rutin and significant heteronuclear multiple bond coherence correlations of NaR.

Fig. 2. NaR ameliorates learning and memory deficits and reduces synaptic loss in APP/PS1 mice.

(A) Timeline of NaR treatment in APP/PS1 mice. (B) Escape latency to the platform during the training trails in a Morris water maze. (C) Target (platform) entries in the probe test. (D) Time spent in target quadrant in the probe test. (E) Mean swimming speed of mice. (F) Representative track images of mice in the probe test. (G) Representative images of dendritic spines in CA1 hippocampal neurons. Scale bar, 5 μm. (H) Quantification of different types of spine density, including thin, stubby, mushroom, and total (n = 22 to 31 from three mice per group). Data are means ± SEM. *P < 0.05 and **P < 0.01, two-way (B) or one-way (C to E and H) analysis of variance (ANOVA), followed by Tukey’s multiple comparisons test. N.S., not significant.

Fig. 3. NaR reduces Aβ deposition but does not alter APP processing.

(A) Representative images of Aβ (6E10) staining in the PFC and hippocampal DG region. (B) Quantification of Aβ plaques in the PFC (n = 13 to 14 slices from three mice per group) and hippocampal DG region (n = 14 to 18 slices from three mice per group). ND, not determined. (C) The amount of soluble and insoluble Aβ fractions extracted from PFC was examined by Western blot analysis. (D) Quantification of the lanes’ intensity with ImageJ software (n = 6 mice per group). (E and F) Expression levels of APPfl, sAPPα, CTFα, CTFβ, BACE, nicastrin, PEN2, and PS2 in PFC were examined by Western blot and quantified with ImageJ software (n = 6 mice per group). Data are means ± SEM. **P < 0.01 and ***P < 0.001, one-way ANOVA, followed by Tukey’s multiple comparisons test.

Fig. 4. NaR enhances microglial phagocytosis of Aβ in vivo and in vitro.

(A) Representative images of Aβ (6E10) plaques and microglia (Iba1) costaining in the cortex from APP/PS1 and NaR-treated APP/PS1 mice. (B) Quantification of microglial cells within 20 μm of the plaque surface (n = 107 to 144 plaques per group). (C) Plaques were divided into small, medium, and large according to their size, and the number of microglia per plaque was quantified. (D) Representative images of Aβ (6E10) plaques, microglia (Iba1), and phagosome (CD68) costaining in the cortex from APP/PS1 and NaR-treated APP/PS1 mice. (E and F) Quantification of the percentage of phagosome area and internalized Aβ using ImageJ Pro Plus software (n = 10 per group). (G) Confocal analysis of microglial phagocytosis of FITC-Aβ₄₂ in the presence or absence of NaR after uptake for 4 hours in cultured primary microglia; CytoD served as a negative control. (H) Quantification of internalized FITC-Aβ₄₂ using ImageJ software (n = 48 to 60 per group). a.u., arbitrary units. (I) FITC-Aβ₄₂ uptake index in the presence or absence of NaR after uptake for the indicated time in cultured primary microglia (n = 6 per group). Data are means ± SEM. *P < 0.05, **P < 0.01, and ***P < 0.001, Student’s t test.

Fig. 5. NaR treatment increases the expression and recycling of phagocytic receptors.

(A) Representative FACS plots and gating strategy to isolate microglia from the brain of adult mice. PE, phycoerythrin. SSC-A, Side-scattered-area; FSC-A, Forward-scattered-area. (B) Relative mRNA levels of phagocytic receptors in sorted microglia from APP/PS1 mice treated with or without NaR (n = 4, 15-month-old mice were treated with or without NaR starting from 13 months of age). (C) Representative images of Aβ (6E10) plaques, microglia (Iba1), and Trem2 costaining in the cortex from APP/PS1 and NaR-treated APP/PS1 mice. (D) Quantification of Trem2 expression level in microglia (APP/PS1, n = 55; APP/PS1 + NaR, n = 63). (E and F) Trem2 recycling assay of microglial BV2 cells in the presence or absence of NaR (n = 13 to 14 per group from three independent experiments). (G) FITC-Aβ₄₂ uptake index after uptake for the indicated time in WT and Trem2 KO cultured primary microglia treated with or without NaR (n = 4 per group). (H) Representative images of thioflavin S staining in the PFC and hippocampal DG region. (I) Quantification of the numbers and areas of Aβ plaques (n = 9 to 12 brain slices from three mice per group). Data are means ± SEM. *P < 0.05, **P < 0.01, and ***P < 0.001, Student’s t test (B, D, and F) or two-way (G) or one-way (I) ANOVA, followed by Tukey’s multiple comparisons test.

Fig. 6. NaR enhances energetic metabolism by promoting mitochondrial OXPHOS in microglia.

(A) Total ATP production of primary microglia under different conditions as indicated. Aβ and/or NaR was added to the cells for 24 hours before measurement (n = 6 from three independent experiments). (B) Schematic diagram of the glycolytic pathway and the mitochondrial TCA cycle, two major pathways of ATP production. (C and D) ECAR measurements of primary cultured microglia in the presence or absence of NaR treatment for 24 hours. Oligomycin (Oligo), an inhibitor of ATP synthase; 2-deoxyglucose (2-DG), a glucose analog. (E and F) OCR measurements of primary cultured microglia in the presence or absence of NaR treatment for 24 hours. p-Trifluoromethoxy carbonyl cyanide phenylhydrazone (FCCP), the reversible inhibitor of OXPHOS; Rote/AA, the mitochondrial complex I and complex III inhibitor. (G) Total ATP production of primary microglia under different conditions as indicated. Galactose (10 μM)–replaced glucose medium was used to incubate cells for 24 hours, and Rote/AA (0.5 μM) was added to the cells (glucose medium) 2 hours before measurement (n = 5). (H) FITC-Aβ₄₂ uptake index after uptake for 4 hours under different cultured conditions as indicated. Galactose-replaced glucose medium was preincubated for 24 hours, and Rote/AA was preadded to the cells (glucose medium) 2 hours before adding FITC-Aβ₄₂ (n = 5). (I and J) The values of ECAR and basal OCR of activated primary microglia induced by LPS (1 μg/ml) or IL-4 (1 μg/ml) with or without NaR treatment. Data are means ± SEM. *P < 0.05, **P < 0.01, and ***P < 0.001, Student’s t test (D and F) or one-way ANOVA, followed by Tukey’s multiple comparisons test (A, C, and G to J).