1.Effcts Of MKP-1 On Synaptic And Cognitive Functions Via Inactivating MAPK/ERK Signaling In AD Mice 2.Effects Of Ginsenoside Rf On AΒ-induced Neurotoxicity

PART I EFFCTS OF MKP-1 ON SYNAPTIC AND COGNITIVE FUNCTIONS VIA INACTIVATING MAPK/ERK SIGNALING IN AD MICEObjective: MAPK was involved in the Aβ deposition,hyperphosphorylation of tau,apoptosis of neurons and so on in the development of Alzheimer’s disease(AD).However,MKP-1 could inactive MAPK,and the pathogenesis of MKP-1 was unclear in AD.In this study,we investigated whether MKP-1 contributed to AD pathogenesis and the underlying mechanism.Methods: 6-month old APP/PS1 double transgenic mice were injected with 1μl of MKP-1 overexpression or sh RNA Adeno-associated virus per site into the lateral ventricle.1.Behavioral test: after 6 months,Morris water maze was used to detect the learning and memory of mice.2.Western blotting: after the behavioral experiment,the expression of the MKP-1,APP,BACE1 and PS1 and the shear fragments C89 and C99 were detected.3.Real-time fluorescence quantitative PCR: Q-PCR detected the APP and BACE1 and MKP-1 m RNA levels.4.ELISA detected the generation of Aβ40 and Aβ42.5.Transmission electron microscopy: after fixation of glutaraldehyde perfusion,the synaptic structure of the hippocampal CA1 area was detected by electron microscope.6.Electrophysiology: the patch clamp record was used to detect LTP in the hippocampal CA1 area.7.Luciferase reporter gene: the effect of MKP-1 and MAPK on the promoter activity of APP and BACE1 were detected by luciferase.Results: Compared with wild type,MKP-1 m RNA and protein expression level were decreased in the brain of AD.Over expression of MKP-1 significantly rescued spatial learning and memory in the Morris water maze and ameliorated hippocampal LTP induction.Transmission electron microscopy indicated that MKP-1 increased the number of synapses in hippocampal.Luciferase reporter gene and Western blot showed overexpression of MKP-1 could decrease APP and BACE1 expression by reducing APP and BACE1 gene transcription and expression.The modulation of APP and BACE1 gene expression by MKP-1 was dependent on ERK.Conclusion: In this study,expression of MKP-1 was decreased with the AD development,and overexpression of MKP-1 rescued hippocampus-dependent behavioral deficits,hippocampal synaptic structure and plasticity of AD mice.Meanwhile,MKP-1 could reduce Aβ and plaque deposition by inhibiting the APP and BACE1 promotor activity and their m RNA level in APP/PS1 double transgenic mice.These results suggested that MKP-1 could regulate synaptic and cognitive functions via inactivating MAPK/ERK signaling in APP/PS1 mice and may be a potential therapeutic target against AD in clinic.PART II EFFECTS OF GINSENOSIDE RF ON Aβ-INDUCED NEUROTOXICITYObjective: Ginseng root as a traditional Chinese medicine has a history of more than 2000 years,and ginsenoside Rf,a kind of ginseng root extract,exerts anti-inflammatory activities in inflammatory responses.Inflammation is one of the main reasons for the AD,thus the purpose of this research is to investigate whether Rf can alleviates the process of the pathogenesis of AD through anti-inflammatory.Methods: The N2A-APP cells and Aβ-induced N2 A cells were treated with Rf1.Western blot : The cell lysate were collected to detect the expression of caspase-1 and caspase-3 associated with inflammation reaction,cell apoptosis and APP-processing,including APP,BACE1,PS1 and degradation of Aβ,such as NEP and IDE.2.ELISA: Cell lysate were collected to detect the Aβ40 and Aβ42.3.Flow cytometry: Flow cytometry was detected the intracellular Ca2+,mitochondrial membrane potential and reactive oxygen species.4.Real-time fluorecence quantitative: Q-PCR was used to detect the inflammatory correlation factor,such as IFN-γ and IL-13.Results: Compared with N2 A cells,the cell death and inflammation were significantly increased with Aβ treatment,such as the increase of intracellular Ca2+,ROS,IFN-γ,caspase-1 and caspase-3 and the decrease of MMP and IL-13.However,Rf could restore these Aβ-induced apoptosis and inflammtion reaction.Meanwhile,Rf could reduce the Aβ level by accelerating the Aβ clearence.Conclusion: Taken together,Rf is able to exert neuroprotective effects against Aβ-induced neurotoxicity through anti-inflammation reaction and anti-oxidative stress,suggesting that Rf may serve as a promising novel therapeutic agent for the prevention and treatment of AD.