The Protective Effect Of Caspr Against Aβ-induced Neurotoxicity

Chapter 1.Caspr causes inhibition of Aβ generation via downregulating APP expressionObjective:To explore the relationship between Caspr(Contactin associated protein)and β-amyloid precursor protein(APP)in the animal model of Alzheimer’s disease and to study the mechanism of Caspr inhibiting the generation of β amyloid(Aβ),in this way to reveal the possible role of Caspr in the pathological mechanism of Alzheimer’s disease.Methods:APP/PS1 transgenic knockout mice were used as an animal model forAlzheimer’s disease research.In the brain of APP/PS1 senile dementia model mice,the distribution of Caspr was observed by immunofluorescencetechnique,the expression of APP protein was recognized by antibody MAB348,β-Actin as an internal parameter.To further understand the effect of Caspr on APP,compared with wild-type mice,the expression level of Caspr protein was measured by Western blotting.EGFP caspr and its empty vector were transfected into HEK-APP cells.After 48 hours,the supernatant was collected for ELISA detection and quantitative analysis of Aβ40,Aβ42 and Aβ42/Aβ40.The expression of caspr in HEK-APP cells was analyzed by ELSIA technique Aβexpression level,to investigate the effect of Caspr on Aβ expression influence.Caspr and empty plasmids were transiently transferred into HEK-APP cells,and protein was extracted 24h later.Western blot was used to detect APP protein expression and quantitative analysis of APP protein and CTFs protein.Using GADPH as internal reference,HEK-293 cells were transfected with EGFP-Caspr and its empty vector.MRNA was extracted 48 hours later and detected by qPCR using APP and Casprprimers.Results:The results showed that Caspr and Aβ were distributed evenly outside the nucleus,and Caspr was highly concentrated around Aβ.In the cerebral cortex of APP/PS1 mice,Caspr was highly located around Aβ.Caspr had no effect on APP mRNA expression level(P>0.05).Compared with the vector group,the expression of APP protein extracted from the cells transfected with Caspr was significantly lower than that of the control group(P<0.05).Compared with the vector group,the expressions of Aβ40 and Aβ42 in the supernatant of cells transfected with Caspr were significantly decreased(P<0.05),while the ratio of Aβ42/Aβ40 in two groups showed no significant difference(P>0.05).Conclusion:Caspr accumulated around APP in the cortex of APP/PS1 model mice.Overexpression of Caspr reduced the expression of APP protein through posttranscriptional regulationbyinhibiting the generation of Aβ.The overexpression of Caspr did not affect y secretase activity,but was involved in APP metabolism.Overexpression of Caspr did not regulate APP metabolism by affecting APP transcription level,but affected exogenous APP and its enzymatic hydrolysate CTFs,further inhibiting the production of Aβ.Caspr may be involved in the pathological mechanism of Alzheimer’s disease.Chapter 2.The protective effect of Caspr against β-induced neurotoxicity via activation of the Akt/Bad pathway and inhibition of apoptosisObjective:By elucidating the neuroprotective effect of Caspr on Aβ-induced neurotoxicity in HEK-293 cells and exploring the mechanism of Caspr involved in Aβproduction.To further investigate whether Caspr has a protective effect on Aβ-induced neurotoxicity by regulating apoptosis pathway under the condition of Aβ-induced neurotoxicity at the cellular level,and to explore the mechanism of Caspr in reversing Aβinducedneurotoxicity.Methods:HEK-293 cells were treated with different concentrations of Aβ42 for 24h after being pre-transfected with Caspr or control plasmid by liposome transfection method,the cell survival status of HEK-293 cells after being treated with different concentrations of Aβ42 was observed.The effect of Aβ42 on HEK-293 cell viability was detected by tetramethyl azazole salt colorimetry,and the cell survival rate was calculated.To further confirm the protective effect of Caspr on HEK-293 cell damage induced by Aβ42,after pre-transfection with Caspr or control plasmid,cells were treated with 10μlAβ42 for 24h,and apoptosis rate was detected by Annexin V/propidium iodide(PI)double staining.In order to further study the potential signal pathway of Caspr mediated neuroprotection,caspase-3,caspase-9,PARP,Bax and Bcl-2 in apoptosis pathway were measured by Western blot,and the ratio of Bcl-2/Bax was calculated.To investigate the effects of Caspr on Akt and Bad phosphorylation,HEK-293 cells were transfected with Caspr to observe the effects of Caspr on Akt and Bad phosphorylation.To further confirm the key role of Akt phosphorylation in Caspr-mediated anti-apoptosis,HEK-293 cells pretransfected with Caspr or control plasmid were compared with Aβ after incubation for 24 hours,LY294002(Akt phosphorylation inhibitor)was treated for 6 hours,and the levels of p-Akt and p-Bad were detected by Western blot.Results:After 24h treatment with Aβ42,the morphologic distortion and contraction of HEK-293 cells occurred in the control group,while the pre-transfection with Caspr significantly inhibited neural death.MTT results showed that the survival rate of HEK-293 cells treated with different doses of Aβ42 decreased significantly with the increase of Aβ42 dose(P<0.05).In addition,compared with the control group,transfection of Caspr significantly increased the survival rate of HEK-293 cells treated with Aβ42(P<0.05).The apoptotic rate of HEK-293 cells treated with Aβ42 increased,while Caspr significantly inhibited the apoptotic effect.The cleavage levels of caspase3,caspase9 and PARP were significantly increased after Aβ42 treatment.However,Caspr pretreatment significantly inhibited the expression levels of caspase-9,caspase-3 and PARP(P<0.05).After Caspr treatment,Bax expression in HEK-293 cells was significantly inhibited,Bcl-2 level was(?)eneased(P<0.05),and Bcl-2/Bax ratio was increased.Protein electrophoresis showed that Caspr overexpression significantly increased Akt phosphorylation(P<0.05),which inhibited Bad phosphorylation,compared with the Aβ42 group.After LY294002 treatment,the level of p-Akt decreased and the level of p-Bad increased,while the expression of caspase-3/-9 and PARP increased significantly under the same conditions(P<0.05).These results suggest that Caspr may prevent Aβ 42-induced HEK-293cell death by activating the Akt/Badanti-apoptotic pathway.Conclusion:Caspr has a neuroprotective effect against Aβ42-induced HEK-293 cytotoxicity by activating Akt/Bad signaling pathway.Caspr antagonizes novel mechanisms of Aβ42-induced apoptosis by enhancing Akt/Bad phosphorylation and inhibiting mitochondria mediated signaling apoptosis pathways.