Rutin prevents tau pathology and neuroinflammation in a mouse model of Alzheimer's disease

Abstract

Background: Tau pathology is a hallmark of Alzheimer's disease (AD) and other tauopathies. During disease progression, abnormally phosphorylated forms of tau aggregate and accumulate into neurofibrillary tangles, leading to synapse loss, neuroinflammation, and neurodegeneration. Thus, targeting of tau pathology is expected to be a promising strategy for AD treatment.

Methods: The effect of rutin on tau aggregation was detected by thioflavin T fluorescence and transmission electron microscope imaging. The effect of rutin on tau oligomer-induced cytotoxicity was assessed by MTT assay. The effect of rutin on tau oligomer-mediated the production of IL-1β and TNF-α in vitro was measured by ELISA. The uptake of extracellular tau by microglia was determined by immunocytochemistry. Six-month-old male Tau-P301S mice were treated with rutin or vehicle by oral administration daily for 30 days. The cognitive performance was determined using the Morris water maze test, Y-maze test, and novel object recognition test. The levels of pathological tau, gliosis, NF-kB activation, proinflammatory cytokines such as IL-1β and TNF-α, and synaptic proteins including synaptophysin and PSD95 in the brains of the mice were evaluated by immunolabeling, immunoblotting, or ELISA.

Results: We showed that rutin, a natural flavonoid glycoside, inhibited tau aggregation and tau oligomer-induced cytotoxicity, lowered the production of proinflammatory cytokines, protected neuronal morphology from toxic tau oligomers, and promoted microglial uptake of extracellular tau oligomers in vitro. When applied to Tau-P301S mouse model of tauopathy, rutin reduced pathological tau levels, regulated tau hyperphosphorylation by increasing PP2A level, suppressed gliosis and neuroinflammation by downregulating NF-kB pathway, prevented microglial synapse engulfment, and rescued synapse loss in mouse brains, resulting in a significant improvement of cognition.

Conclusion: In combination with the previously reported therapeutic effects of rutin on Aβ pathology, rutin is a promising drug candidate for AD treatment based its combinatorial targeting of tau and Aβ.

Keywords: Alzheimer’s disease; Neurofibrillary tangles; Neuroinflammation; Rutin; Synapse loss; Tau pathology.

Fig. 1

Rutin inhibits tau aggregation, reduces tau oligomer-induced cytotoxicity and proinflammatory cytokine production. A The chemical structure of rutin. B The aggregation kinetics of 10 μM tau was assessed by thioflavin T fluorescence assay with or without the incubation of 40 or 80 μM of rutin. C The morphologies of tau were examined using a Hitachi H7650 TEM (scale bars 10 μm). D The viability of SH-SY5Y cells was determined by MTT assay when challenged with 1 μM tau oligomers in the presence or absence of rutin. E The levels of IL-1β and TNF-α were detected in the supernatants of primary microglial cultures when challenged with 1 μM tau oligomers in the presence or absence of 8 μM rutin. Experiments B, D, E were performed three times with at least biological triplicates in each experiment. Data represent means ± SEM and were analyzed by two-way ANOVA with Bonferroni’s test (B) or one-way ANOVA with Tukey’s test (D, E). **P < 0.01, ****P < 0.0001. ns, not significant

Fig. 2

Rutin protects neuronal morphology from toxic tau oligomers and promotes microglial engulfment of extracellular tau. A Immunolabeling of MAP2 (green) in primary neuronal cultures treated with 1 μM tau oligomers in the presence or absence of 8 μM rutin for 24 h (scale bar: white, 25 μm; red, 10 μm). B The concentric rings spaced 20 μm apart centered on the soma center, which was used to count the number of intersections for the Sholl analysis. Scale bar: 40 μm. C Sholl analysis of the dendritic branching in primary neuronal cultures treated with 1 μM tau oligomers in the presence or absence of 8 μM rutin for 24 h. Forty neurons per culture from three independent cultures were used for the analysis. D Immunolabeling of tau (green) within Iba-1⁺ (red) cell in primary microglial cultures treated with 1 μM tau oligomers in the presence or absence of 8 μM rutin for 24 h (scale bar: 10 μm). E Quantification of tau puncta engulfed by Iba-1⁺ microglial cells in D. At least 20 microglial cells per culture from three independent cultures were used for the analysis. Data represent means ± SEM and were analyzed by two-way ANOVA with Bonferroni’s test (C) or Student’s t test (E). ***P < 0.001

Fig. 3

Rutin rescues memory deficits in Tau-P301S mice. A Schematic representation of pharmacological treatment and experimental measurement. B–D The Morris water maze was performed on Tau-P301S mice and their WT littermates treated with rutin or vehicle. (n = 8 mice). B The latency to find the hidden platform was measured during training trials. C, D During probe trials, the latency to the position of the removed platform (C) and the number of platform crossings (D) were measured. E The time spent in the novel arm in Y-maze test was determined on Tau-P301S mice and their WT littermates treated with rutin or vehicle (n = 8 mice). F The novel object recognition test was performed on Tau-P301S mice and their WT littermates treated with rutin or vehicle. The results were expressed as the discrimination index (n = 8 mice). Data represent means ± SEM and were analyzed by two-way ANOVA with Bonferroni’s test (B) or one-way ANOVA with Tukey’s test (C–F). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ns, not significant

Fig. 4

Rutin attenuates tau pathology in Tau-P301S mice. A AT8 immunostaining for phosphorylated-tau in the brains of Tau-P301S mice and their WT littermates treated with rutin or vehicle (scale bar: 100 μm). B, C Quantification of AT8 immunostaining in the hippocampal CA1 (B) and DG (C) regions (n = 8 mice). D Western blot analysis of phosphorylated-tau (AT8), PP2A, and β-actin in the brain lysates of Tau-P301S mice and their WT littermates treated with rutin or vehicle. E, F Quantification of AT8 positive p-tau (E) and PP2A (F) in D (n = 8 mice). G Dot-blot analysis of OC-positive oligomers in the brain lysates of Tau-P301S mice and their WT littermates treated with rutin or vehicle. H Quantification of OC-positive tau oligomers (n = 8 mice). Data represent means ± SEM and were analyzed by one-way ANOVA with Tukey’s test (B–F, H). ***P < 0.001, ****P < 0.0001, ns, not significant

Fig. 5

Rutin reduces neuroinflammation in the brains of Tau-P301S mice. A GFAP immunostaining and Iba-1immunostaining in the brains of Tau-P301S mice and their WT littermates treated with rutin or vehicle (scale bar: black, 100 μm; red, 10 μm). B, C Quantification of GFAP (B) and Iba-1 (C) immunostaining in A. (n = 8 mice). D, E The levels of IL-1β (D) and TNF-α (E) in the brain lysates of Tau-P301S mice and their WT littermates were determined using corresponding ELISA kits. (n = 8 mice). F Western blot analysis of GFAP, Iba-1, and β-actin in the brain lysates of Tau-P301S mice and their WT littermates treated with rutin or vehicle. G Quantification of GFAP and Iba-1 in F. (n = 8 mice). H Western blot analysis of p-P65, total P65, IKKβ, and β-actin in the brain lysates of Tau-P301S mice and their WT littermates treated with rutin or vehicle. I Quantification of p-P65/P65 and IKKβ in H. (n = 8 mice). Data represent means ±SEM and were analyzed by one-way ANOVA with Tukey’s test (B–E, G, I). **P < 0.01, ***P < 0.001, ****P < 0.0001, ns, not significant

Fig. 6

Rutin rescues synapse loss in Tau-P301S mice. A PSD95 immunostaining and synaptophysin immunostaining in the brains of Tau-P301S mice and their WT littermates treated with rutin or vehicle (scale bar: 3 μm). B–D Quantification of PSD95 puncta (B), synaptophysin puncta (C) and their apposition (D) in A. (n = 8 mice). Data represent means ± SEM and were analyzed by one-way ANOVA with Tukey’s test (B–D). **P < 0.01, ****P < 0.0001, ns, not significant

Fig. 7

Rutin prevents microglial synapse engulfment in Tau-P301S mice. A Representative images show the engulfed PSD95 (red) puncta within Iba-1⁺ (green) microglial cells in the brains of Tau-P301S mice and their WT littermates treated with rutin or vehicle (scale bar: white, 10 μm; cyan, 5 μm; red, 5 μm). B Quantification of PSD95 puncta per Iba-1⁺ microglial cell (n = 8 mice). Data represent means ± SEM and were analyzed by one-way ANOVA with Tukey’s test. ****P < 0.0001, ns, not significant