Se-yeast Can Improve The Clearance Of A? And Tau In AD By Regulating Autophagy Pathway

Objective: Alzheimer's diseases(AD) is an age-related neurodegenerative diseases often occurring in the elderly. Senile plaques(SP) and neurofibrillary tangles(NFT) in the brain are the two major neuropathological hallmarks of AD. It was now believed that accumulated denatured proteins such as A? and phosphorylated Tau can not be effective cleared from neurons might accelerate the pathologenesis of AD. Autophagy is an intracellular degradation pathway for disposal of denatured proteins or damaged organelles to maintain intracellular homeostasis. It was regarded as one of the major pathway to remove A? and phosphorylated Tau. Thus autophagic dysfunction is considered as a major cause of AD. Modulation of autophagy could be a new target and direction in treatment of Alzheimer's disease. Selenium yeast is rich in organic selenium, 70% of selenium in the yeast is present in the form of selenomethionine(Se-Met). In this study we explore the effect and mechanism of Selenium yeast about clearing A? and Tau by modulating autophagy in triple transgenic(Psen1, APPSwe and tauP301L)(3×Tg) AD mice. Moreover, in vitro, primary neuron and N2a-APP695-sw are treated with Se-Met to explore the therapeutic effect of AD and the underlying mechanism, which is expected to provide a scientific basis for the new AD drugs exploration.Methods: 1)In vivo: 4-month-old 3×Tg AD mice were fed fodder with 5.18g/Kg Selenium yeast. After treatment for 4 months, the cognitive ability was detected by the morris water maze and step-down test; Atomic Fluorescence Spectrophotometer was uesed to analysis selenium content of brain and internal organs in mice; Level of oxidative stress(SOD), lipid peroxidation(MDA) and activity of selenoenzymes(TrxR?Gpx)were measure by specific Kits; Immunofluorescence and western-blot were used to test the changs of following proteins: A oteinsg sed to test LC3, p62, signal transduction proteins of autophagy process(Akt?mTOR?p70s6K?AMPK?cathepsinD(Cat-D)); Autophagosomes subpopulation were isolated by differential centrifugation from brain tissue and the contents of A?, Tau, Tau-ps202 and Tau-ps422 were detected by western-blot. 2)In vitro: Primary neuron were isolated from newborn mice(WT, 3×Tg) and cultured for 7 days before treated with 1?mol/L Se-Met for 24 h. Immunofluorescence, Western-Blot were used to evaluate the changs of following targets: A?, APP, BACE-1, Tau, LC3, p62, Akt, mTOR, p70s6 K, AMPK, Cat-D; N2a-APP695-sw cells were also treated with 1?mol/L Se-Met for 24 h, and the expression levels of A?, APP, BACE-1, Tau, LC3, p62, Akt, mTOR, p70s6 K, AMPK, Cat-D in the cells were detected by Western blot and Immunofluorescence.Results: 1) In vivo, the escape latency of selenium yeast treated mice was significantly shorten than control(++p<0.01); Times of crossing and the time spent in the quadrant of original platform located were significantly increased in 24 h probe trial(*p<0.05); During the step-down test, t, the error numbers of selenium yeast treated mice significantly decreased compare to those of the control mice in the 3rd training day(*p<0.05), what's more, the average step-down latency was longer than that of the control mice in the 24h-memory testing; The selenium content in different tissues of AD mice were much lower than in those of WT mice, after the treatment of selenium yeast, the levels of selenium in most of those tissues recovered significantly(*p<0.05, **p<0.01). Moreover, the expression of APP, A? oligomer and BACE-1 all significantly decreased after the treatment of selenium yeast(**p<0.01,*p<0.05). Immunohistological study also showed the level of total Tau and A?1-42 were significantly reduced as well(**p<0.01,*p<0.05). Furthermore, the expression levels of LC3 in both hippocampus and cortex significantly decreased compared to the control(**p<0.01), while the colocalization of LC3 and Tau in hippocampus significantly increased(*p<0.05). The expression of Akt, P-Akt, mTOR, p70s6 K as well as P-p70S6 K dramaticly increased(**p<0.01,*p<0.05), while the expression of AMPK and P-AMPK significantly decreased(**p<0.01,*p<0.05), the level of the adaptor molecule p62 also decreased while the Cat-D was increased after treating with selenium yeast. In the subcomponents of autophagosome, the expression levels of A?, Tau, Tau-ps202 and Tau-ps422 in autophagosomes from AD mice(Tg) were much higher than those from WT mice, after treatment of selenium yeast, the levels of those proteins significantly decreased; while in autolysosomes, an reversal tendency was observed, the expression levels of A?, Tau, Tau-ps202 and Tau-ps422 were much lower in Tg micethan WT mice, and selenium yeast treatment obviously increased the levels of these proteins. The activity of SOD increased while the level of MDA significantly decreased in the brain of selenium yeast treated mice), the activity of selenium-enzyme(Trx R?Gpx) also significantly increased(**p<0.01,*p<0.05).2)In vitro: Compared with WT primary neurons, the expression of APP, A?, BACE-1, LC3 in 3×Tg mice significantly increased(##p<0.01, ###p<0.001), after Se-Met treated 24 h, the expression of APP, A ?, BACE-1, LC3 significantly decreased(**p<0.01, *p<0.05,***p<0.001). Immunohistochemistry showed that Se-Met can promot colocalization of LC3 and Tau. After Se-Met treated 24 h, the expression levels of Akt and P-Akt were obviously increased, while, mTOR, p70s6 k and their phosphorylation decreased after Se-Met treated 24h(**p<0.01,*p<0.05) at the same time, Beclin1 and P62 decreased, Cat-D increased(*p<0.05). In N2a-APP695-sw cell experiments, APP, A? oligomer, ?-CTF and BACE-1 were significantly lower than control(**p<0.01,*p<0.05) after Se-Met treated 24 h. Meanwhile the level of LC3 decreased(*p<0.05).The levels of Akt, mTOR, p70s6 k and their phosphorylation were obviously increased, P62, Beclin1 were decreased(**p<0.01,*p<0.05), Cat-D increased(**p<0.01) after Se-Met treated. What's more, Se-Met can promot colocalization of LC3 and A ?, and it can promote autophagosome changing into autolysosome detected by fluorescence probe named mTagRFP-mWasabi-LC3.Conclusion: Selenium yeast can significantly improve the cognitive ability of 3×Tg AD mice, decrease levels of A?, APP, BACE1, and tau in the brain, restore the selenium content in AD mice, enhance the antioxidant capacity.Our study proved that selenium yeast(in vivo) or selenomethionine(in vitro) could decrease the level of A? and Tau by both inhibit the generation of A? and promote the clearance of A? and Tau through modulating the autophagy signaling pathways. Therefore, selenium yeast has real potential in AD therapy.