The Study Of The Protective Role Of Sphingosine Kinase-1on The APP/PS1Double Transgenic Mouse Model Of Alzheimer’s Disease

Part I. Overexpression of Sphingosine Kinase-1Rescues Transgenic Mouse Model of Alzheimer’s Disease from Neurotoxicity of Amyloid-β (A(3) proteinObjective:Aberrant production and accumulation of amyloid-β (A(3) protein is central t the pathogenesis of Alzheimer’s disease (AD) and has a causal role in dementia. Evidenc showed that imbalance of sphingosine1-phosphate (SlP)/ceramide rheostat may be part o a mechanism. The aim of this study was to explore the effects of overexpression o sphingosine kinase-1on the APP/PS1transgenic mouse model of Alzheimer’s Disease.Methods:In this study, APPsw/PS1double-transgenic (APP/PS1) mice were adopted an received an intra-hippocampal injection of recombinant adenovirus-mediated vector targeting Sphkl gene(rAV-Sphkl) to evaluate the role of sphingosine kinase-1(Sphkl), critical regulator of the balance between SIP/ceramide rheostat, in the pathologica progression of AD. Confocal microscopy was used to evaluate the disposition of amyloi protein. The protein expression of sphklwere determined by Western blot. Elisa wa designed to assess the secretion of amyloid protein. Mirror water maze was used to detec the damage of behavior of APP/PS1double transgenic mouse model.Results:Administration of rAV-Sphkl to the APP/PS1mice four weeks later exhibite overexpression of Sphkl protein coupled with decreased deposition of Aβ protein follo we*by improved learning and memory abilities. Conclusions:These results demonstrate that overexpression of Sphkl could efficiently attenuate the pathological damage of AD. Therefor, Sphkl overexpression mitigated the pathological damage of AD, establishing a critical role of Sphkl overexpression for AD and Sphkl gene delivery would be a novel strategy for the treatment of AD. Part II. The mechanism of the protective effects of sphingosine kinase1on the of APP/PS1double transgenic AD miceObjective:The first part of the study found that sphingosine kinase1over-expression can reduce the deposition of senile plaques in mice and mitigate the damage of their behavior, but we have not yet to clear the way of Sphkl protective effect. The purpose of this part is to explore the mechanism of the protective effects of sphingosine kinase1on the APP/PS1double transgenic AD mice.Methods:In this section, Caspase-3protein expression was assessed by Western blot. TUNEL was used to assay the degree of apoptosis. The expression of slprl, slpr3mRNA were measured by real-time PCR. Enzyme-linked immunosorbent assay (ELISA) designed to evaluate the CER, SIP secretion. Ultrafine electron microscopy to detect the hippocampal ultEastructure of APP/PS1double transgenic mouse.Results:Four weeks after stereotactic injection, the APP/PS1mice showed a decline in ceramide/SlP proportion coupled with a lower degree of apoptosis. And Sphkl over-expression could inhibite the caspase-3expression significantly. Elevated SlPrl expression and postsynaptic membrane SNAP-25protein expression increased synaptic activity.Conclusion:These findings suggest that overexpression of Sphkl could suppress apoptosis via the capases-3pathway and up-regulate synaptic activity by SNAP-25and slp receptor (slp-GPCRs). Part Ⅲ. Effects of Small Interfering RNA Targeting Sphingosine Kinase-1Gene On The Animal Model Of Alzheimer’s DiseaseObjective:Alzheimer’s disease (AD) is an age-related, progressive neurodegenerative disorder that occurs gradually and results in memory, behavior, and personality changes. Abnormal sphingolipid metabolism was reported in AD previously. And our previous study showed that:over-expression of Sphkl could suppress apoptosis and relieve the deposition of Aβ protein,which demonstrated from the good side the protective role of Sphkl in the APP/PS1double transgenic mouse.The aim of this study was to explore the effects of downregulating the expression of Sphkl expression by the way of small interfering RNA(siRNA) on the APP/PS1double transgenic mouse.Mehtods:An adenovirus vector that expressed small interfering RNA(siRNA)against the Sphkl gene (Sphk1-siRNA) was designed, then effects of Sphk1-siRNA on the APP/PS1mouse4weeks after treatment with sphk1-siRNA hippocampcal injection was examined. Sphkl protein expression were confirmed using Western blot and ceramide content coupled with S1P secretion was evaluated by enzyme-linked immunosorbent assay (Elisa). AP load was detected by Immunohistochemical staining and ELISA. Morris water maze was adopted to test the learning and memory ability of the APP/PS1mice.Results:A significant difference in the expression of Sphkl protein and mRNA was observed between the siRNA group and the control group. Aβ load in transfected mice was accelerated in vivo, with significant aggravation of the learning and memory ability.Conclusion:The results show that the recombinant adenovirus vector containing the sphkl gene was successfully constructed, which can accelerate the burdern of Aβ load and the learning and memory ability, silence SPhK1gene in vivo. The Sphkl gene modulation in the in the animal model of AD may be important for the treatment of AD. Part IV. The mechanism of Small Interfering RNA Targeting Sphingosine Kinase-1Gene On Apoptosis of The Animal Model Of Alzheimer’s DiseaseObjective To observe the effect of the recombinant adenovirus vector that expressed small interfering RNA(siRNA)against the sphkl gene (Sphkl-siRNA) on the apoptosis of APP/PS1double transgenic mouse.Methods After the recombinant adenovirus vector containing the sphkl gene was successfully constructed, then effect of the recombinant adenovirus vector that expressed small interfering RNA(siRNA)against the sphkl gene (siRNA-sphkl) on the apoptosis of APP/PS1double transgenic mouse30days after treatment with siRNA-sphkl hippocampcal injection was examined.Results:Compared with the saline group, the apoptosis degree and the caspase-3expression in the siRNA-sphkl group were markedly increased.Conclusions The results show that the recombinant adenovirus vector containing the sphkl gene was successfully constructed, which can increase the apoptosis of hippocampus in vivo.