The Effects Of Selenomethionine On Neurogenesis In A AD Mouse Model

Objective:Alzheimer’s disease(AD)is a neurodegenerative disease with the major clinical manifestations of cognitive disorders and memory loss,which might deteriorate overtime.Both animal and clinical researche have implicated neurogenesis dysfunction as an important early event of the pathology of AD,and it was directly correlated to cognitive dysfunction.Pharmacological or genetic induction of neurogenesis can ameliorate neurodegeneration and cognitive deficit as well.Therefore,the induction of neurogenesis is considered as an effective strategy to reduce neurodegeneration and slow disease progression in AD.The majority of Se-Met in tissues is sourced from food,a primary means through which organisms obtain organic Se.In this study,we examined the effects of Se-Met on NSCs proliferation and differentiation as well as its effects on neurogenesis in the brain of 3xTg AD mice,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 water with 6 μg/ml Se-Met.After treatment for 3 months,Immunofluorescence and western-blot were used to test the changs of following proteins:A oteinsg sed to test NeuN,GFAP,DCX and Nestin.Signal pathway transduction proteins of neurogenesis process(PI3K,Akt,GSK3β,β-catenin,Cyclin-D.2)In vitro:Primary neuron were isolated from newborn mice(WT,3×Tg)and cultured for 3 days before treated with flow cytometer.The resulting CD24＋CD133＋NSCs were then collected and cultured for cell proliferation or differen-tiation experiments.To analyze the effects of Se-Met on NSC proliferation,NSCs were plated in black bottom 96-well microplates at a density of 10,000 cells/well,NSCs were treated with Cell Counting Kit-8.Also,a BrdU proliferation assay was also used to evaluate the proliferation of NSCs.After that,Western-Blot were used to evaluate the changs of following targets:PI3K,Akt,GSK3β,β-catenin,Cyclin-D,and so on.Immunofluorescence staining revealed MAP2 for neurons and GFAP for astrocyte.Results:1)In vitro:Primary neuron were isolated from newborn mice(WT,3×Tg).The CCK-8 demonstrated the ability of low-concentration Se-Met(≤10 μM)to promote NSCs proliferation and that of high-concentration Se-Met(≥50 μM)to have an inhibitory effect.An increase in the number of BrdU＋ cells after Se-Met treatment was also observed,suggested that when the number of plated cells remained constant,Se-Met-treated NSCs would yield significantly larger neurospheres than the control NSCs(**p<0.01).In our previous studies,we noticed that Se-Met significantly decreased the activity of glycogen synthase kinase-3β(GSK3β)in the brains of AD mice(*p<0.05).Our results indicated that Se-Met significantly depressed the activity of GSK3b,an enzyme that has an important impact on Wnt signaling pathway.Se-Met treatment substantially elevated b-catenin activity and expression of its downstream protein Cyclin-D(*p<0.05).Cyclin-D can promote mitosis and is pivotal to cell proliferation.Our previous findings also indicated that Se-Met might influence Akt activity.Se-Met treatment tremendously facilitated activities of Akt and PI3K in NSCs(**p<0.01,*p<0.05),which demonstrated that Se-Met elevated NSC proliferation and differentiation by stimulating the PI3K-Akt-GSK3β-Wnt signaling pathway.Our subsequent observations on cultured NSCs discovered that,after neurospheres adhered,their spreading out was substantially accelerated with Se-Met treatment.After 3 weeks of incubation,Se-Met led to an increase in neuronal marker NeuN protein levels,and a decrease in the astrocyte marker GFAP.MAP2 immunofluorescent labeling presented a significant growth in both the number and size of neurons after Se-Met treatment.2)In vivo,we investigated whether Se-Met promoted hippocampal NSC proliferation in A D model mice.After Se-Met treatment,the number of Nestin＋ NSCs dramatically rose in the dentate gyrus region of 3xTg AD mice in comparison with controls.There was a statistically insignificant growth in total PI3K protein levels in hippocampal tissues of Se-Met-treated AD mice.In addition,by contrast,p-PI3K levels and the p-PI3K/PI3K ratio significantly increased(*p<0.05).Akt and p-Akt levels increased moderately compared with controls.Furthermore,the activity of crucial proteins in the Wnt pathway,such as b-catenin and Cyclin-D also increased upon Se-Met treatment(*p<0.05).which demonstrated that Se-Met elevated NSCs proliferation and differentiation by stimulating the PI3K-Akt-GSK3β-Wnt signaling pathway.Conclusion:Our study illustrated that Se-Met potently induced NSCs proliferation and neuronal differentiation both in vitro and in vivo.It exerts treatment effect through modulating the PI3K-Akt-GSK3β-Wnt signaling pathway.The multi-faceted therapeutic effect of Se-Met in AD is worthy of further investigation to thoroughly understand the mechanism.