| Objectives: Alzheimer’s disease(AD) is a chronic progressive neurodegenerative disease in central nervous system. The dysfunction of autophagosomes’ transportion caused by accumulation of autophagosomes in distal of axon interrupt the generation of Aβ and promoted the development of AD. Molecular motors dynein and microtubule-associated protein 2(MAP2) are tightly related with autophagosomes’ transportion and the pathogenesis of AD. GSK-3β plays a key role in regulation of molecular motors, which is a new therapeutic target for treatment of AD. Curcumin is a plant compound extracted from the herb of turmeric rhizome, and can remarkably reduce the generation of Aβ and had neuroprotection in AD, but the mechanism is still unclear. Our study aims to observe autophagosomes and axonal transport of autophagosomes, then observe the effects of Curcumin on the motor protein dynein, MAP2 and the Aβ generation. Furthermore, whether this effect depends on the inhibition of GSK-3β will be studied. Our study will explore the potential mechanisms of neuroprotection of Curcumin from a new perspective and further elucidate the multiple protective mechanism of Curcumin on prevention and treatment of AD.Methods: In vivo: 30 six-month-old APP/PS1 double transgene mice were randomly divided into untreated(the normal diet), low-dose(diet mixed with Curcumin 0.16g/Kg) and high-dose groups(diet mixed with Curcumin 1.0g/Kg), and then all the mice were continuously fed for 6 months. The Morris water maze test was used to study the effects of curcumin on spatial learning and memory of AD mice. IHC were used to detect the expression of Aβ42; the transmission electron microscope was employed to observe the changes of autophagosomes; western blot was used to examine the protein expression of Beclin1, LC-3; IHC were used to detect the expression of Microtuble-associate Proteins 2(MAP2); western blot was used to examine the protein expression of Tau, p-Tau(Ser404), Glycogen Synthase Kinase-3β(GSK-3β), p-GSK-3β(Try216), Dynein intermediate chain(DIC). In vitro: N2a/WT and N2a/APP695 swe were divided into WT, control(DMSO), low-dose(1.25 μM Curcumin) and high-dose(5.0 μM Curcumin) groups. ELISA was used to detect the expression of Aβ42; the transmission electron microscope was employed to observe the autophagosome’s number; western blot was used to analyze the protein level of Beclin1, LC3; Immunofluorescence was used to detect the co-localization of LC3 and Aβ; western blot was used to analyze the protein level of Tau, p-Tau(Ser404), DIC, GSK-3β and p-GSK-3β(Tyr216). The mRFP-GFP-LC3 adenovirus transfected cells were used to observe the autophagosome flux.Results: In vivo: Immunohistochemistry results showed that the area of Aβ42 plaques was significantly reduced in brain in Curcumin-treated group; electron microscopy found that the accumulation of autophagosomes in axon decreased, while autolysosomes in cytoplasm of neuron increased in Curcumin-treated group; western blot showed that Curcumin increased the expression of Beclin1 and LC-3II/I; immunohistochemistry showed that MAP2 increased significantly(p<0.05); western blot showed that Curcumin inhibited the expression of p-Tau(Ser404) / Tau, p-GSK-3β(Tyr216) / GSK-3β expression and increase the expression of DIC. In vitro study: ELISA showed that the dose of Aβ42 decreased after Curcumin treatment(p<0.05); electron microscopy found the number of autolysosomes and autophagosomes was increased after Curcumin treatment; western blot showed that Curcumin increased the protein expression of Beclin1, LC-3II/I, DIC, and inhibited p-Tau(Ser404)/Tau and p-GSK-3β(Tyr216)/GSK-3β; immunofluorescence showed LC3 was colocalized with Aβ; mRFP-GFP-LC3 adenovirus transfect results showed the binding of autophagosomes and lysosomes increased.Conclusion: Curcumin could increase the number of autolysosomes and autophagosomes, increase expression of MAP2, suppress hyperphosphorylated Tau protein and increase synaptic DIC expression and facilitate axonal transport, which are depended on the activity of GSK-3β. |
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