Selenomethionine Mitigates Cognitive Decline by Targeting Both Tau Hyperphosphorylation and Autophagic Clearance in an Alzheimer's Disease Mouse Model

Abstract

Tau pathology was recently identified as a key driver of disease progression and an attractive therapeutic target in Alzheimer's disease (AD). Selenomethionine (Se-Met), a major bioactive form of selenium (Se) in organisms with significant antioxidant capacity, reduced the levels of total tau and hyperphosphorylated tau and ameliorated cognitive deficits in younger triple transgenic AD (3xTg-AD) mice. Whether Se-Met has a similar effect on tau pathology and the specific mechanism of action in older 3xTg-AD mice remains unknown. Autophagy is a major self-degradative process to maintain cellular homeostasis and function. Autophagic dysfunction has been implicated in the pathogenesis of multiple age-dependent diseases, including AD. Modulation of autophagy has been shown to retard the accumulation of misfolded and aggregated proteins and to delay the progression of AD. Here, we found that 3xTg-AD mice showed significant improvement in cognitive ability after a 3-month treatment with Se-Met beginning at 8 months of age. In addition to attenuating the hyperphosphorylation of tau by modulating the activity of Akt/glycogen synthase kinase-3β and protein phosphatase 2A, Se-Met-induced reduction of tau was also mediated by an autophagy-based pathway. Specifically, Se-Met improved the initiation of autophagy via the AMP-activated protein kinase-mTOR (mammalian target of rapamycin) signaling pathway and enhanced autophagic flux to promote the clearance of tau in 3xTg-AD mice and primary 3xTg neurons. Thus, our results demonstrate for the first time that Se-Met mitigates cognitive decline by targeting both the hyperphosphorylation of tau and the autophagic clearance of tau in AD mice. These data strongly support Se-Met as a potent nutraceutical for AD therapy.SIGNIFICANCE STATEMENT Selenium has been widely recognized as a vital trace element abundant in the brain with effects of antioxidant, anticancer, and anti-inflammation. In this study, we report that selenomethionine rescues spatial learning and memory impairments in aged 3xTg-AD mice via decreasing the level of tau protein and tau hyperphosphorylation. We find that selenomethionine promotes the initiation of autophagy via the AMPK-mTOR pathway and enhances autophagic flux, thereby facilitating tau clearance in vivo and in vitro We have now identified an additional, novel mechanism by which selenomethionine improves the cognitive function of AD mice. Specifically, our data suggest the effect of selenium/selenomethionine on an autophagic pathway in Alzheimer's disease.

Keywords: Alzheimer's disease; autophagy; hyperphosphorylated tau; selenomethionine; tau; tauopathy.

Figure 1.

Se-Met administration ameliorates cognitive deficits in 3xTg-AD mice. A–F, The Morris water maze test was used to evaluate spatial memory in mice. A, B, During the 4 d of spatial orientation trials, escape latencies and swimming speed were measured to assess the memory and the locomotor ability of the mice, respectively. C–F, For the 24 and 72 h probe trials, respectively, the time spent in the target quadrant and the number of crossings over the original platform area were recorded. G, H, The latency period and number of errors during a step-down-type passive avoidance test were detected. The values are shown as the mean ± SEM, and 12 mice were tested per group. *p < 0.05; **p < 0.01.

Figure 2.

Se-Met decreases the level of total tau and hyperphosphorylated tau. A–D, Se-Met significantly reduced the levels of total tau and pS404-tau protein in the TBS-soluble and TBS-insoluble fractions of the hippocampus (A, B) and cortex (C, D) in 3xTg-AD mice. E, F, Immunofluorescence images of tau in the hippocampus (DG and CA1) and cortex and pS404-tau in the hippocampus (CA3). G, H, Representative Western blot images and densitometric analysis of total tau and pS404-tau in primary cortical neurons. I, J, Immunofluorescence images of tau and pS404-tau in primary cortical neurons. Quantitative results were normalized against the levels of GAPDH. The data were expressed as percentages of the control (set to 100%) and presented as the group mean ± SEM (n = 6). *p < 0.05; **p < 0.01; ***p < 0.001. Scale bars, 20 and 50 μm. Hipp, Hippocampus; DG, dentate gyrus; CA1, cornu ammonis 1; CA3, cornu ammonis 3.

Figure 3.

Se-Met decreases tau phosphorylation via activation of Akt and inhibition of GSK3β. A, B, Representative Western blot images and quantification of GSK3β, pGSK3β (Ser9), PP2A, p-PP2A (Tyr307), Akt, and p-Akt (Ser473) in the hippocampus of WT, Se-Met-treated, and control mice. C, D, Representative Western blot images and analysis of these kinases in the cortex of WT, Se-Met-treated, and control mice. E, F, Western blot images and analysis of these kinases in primary cortical neurons. Densitometry results were normalized against the levels of GAPDH. The values are expressed as percentages of the control (set to 100%) and presented as the group mean ± SEM (n = 6). *p < 0.05; **p < 0.01.

Figure 4.

Se-Met has no effect on tau P301L transcription and increases the formation of autophagosomes in the hippocampus of 3xTg-AD mice. A, B, The level of tauP301L mRNA in the hippocampus was investigated by RT-PCR. Quantitative results were normalized against the level of actin. Values are expressed as percentages of the control (set to 100%) and presented as the group mean ± SEM (n = 4). C, The double-membrane-bound autophagosomes in the hippocampus of 3xTg-AD mice were observed using transmission electron microscopy. Scale bars, 0.2 μm.

Figure 5.

Se-Met enhances tau clearance by promoting the initiation of autophagy via the AMPK–mTOR pathway. A, B, Representative Western blot images and densitometric analysis of LC3-II, mTOR, p-mTOR (Ser2448), p70S6K, p-p70S6K (Thr389), AMPKα, and pAMPKα (Thr172) expression in the hippocampus of WT, Se-Met-treated, and control mice. C, D, Representative Western blot images and densitometric analysis of these kinases in the cortex of WT, Se-Met-treated, and control mice. E, Immunofluorescence staining was performed to detect the expression and localization of LC3 and total tau in the hippocampus (DG, CA1, and CA3). F, G, Representative Western blot images and densitometric analysis of LC3-II, mTOR, p-mTOR (Ser2448), p70S6K, p-p70S6K (Thr389), AMPKα, and pAMPKα (Thr172) in primary cortical neurons. Densitometry results were normalized against the levels of GAPDH. Values are expressed as percentages of the control (set to 100%) and presented as the group mean ± SEM (n = 6). *p < 0.05; **p < 0.01; ***p < 0.001. Scale bar, 50 μm. DG, Dentate gyrus; CA1, cornu ammonis 1; CA3, cornu ammonis 3.

Figure 6.

Se-Met decreases p62 protein expression and increases cathepsin D protein expression. A–D, Representative Western blot images of p62 and CatD expression in the hippocampus (A, B) and cortex (C, D) of WT, Se-Met-treated, and control mice. E, Immunofluorescence staining of CatD in the hippocampus (DG, CA1, and CA3) and cortex. F, G, Representative Western blot images of p62 and CatD expression in primary cortical neurons. Densitometry results were normalized against the levels of GAPDH. Values are expressed as percentages of the control (set to 100%) and presented as the group mean ± SEM (n = 6). *p < 0.05; **p < 0.01. Scale bar, 50 μm. DG, Dentate gyrus; CA1, cornu ammonis 1; CA3, cornu ammonis 3.

Figure 7.

Se-Met influences the levels of total tau and phosphorylated tau in the autolysosome, autolysosome, and lysosome fractions isolated from the brain of 3xTg-AD mice. A, Autophagosome, autolysosome, and lysosome fractions were isolated from the brain of WT, Se-Met-treated, and control mice. The relative enrichment of autophagic and lysosomal protein markers was detected by Western blot with anti-LC3 and anti-Lamp1 antibodies (top two rows), respectively. The expression of p62, total tau, and phosphorylated tau in these fractions was detected (bottom three rows). B, C, Densitometry analysis of total tau (B) and phosphorylated tau (C) expression in different fractions. CBB, Coomassie Brilliant Blue.

Figure 8.

Se-Met enhances autophagic flux by promoting autophagolysosomal fusion. N2a-sw cells were treated with bafilomycin A1 (50 nm, 4 h) to block the fusion of autophagosomes with lysosomes, and the effect of Se-Met (1 μm) on autophagic flux was assessed. A, B, The expression of p62 and LC3II was detected by Western blot. Quantitative results were normalized against the levels of β-actin. Values are expressed as percentages of the control (set to 100%) and presented as the group mean ± SEM (n = 3). *p < 0.05; **p < 0.01; ***p < 0.001. C, LC3 expression was detected by immunofluorescence staining in N2a-sw cells. D, Representative fluorescent images of N2a-sw cells transiently transfected with mTagRFP-mWasabi-LC3, followed by bafilomycin A1 and/or Se-Met treatment. Scale bars, 10 and 20 μm.