Effect Of TYROBP Gene On Phosphorylation Of Tau Protein In PSEN1 P.G378E Mutation Mice

BackgroundAlzheimer’s disease(AD)is the most common type of dementia among the elderly people.It is too difficult to cure.At present,there are about 50 million AD patients wordwide with increasing rate of every 3 seconds one case.The main pathological features of AD are extracellular senile plaque formed by the deposition of β-amyloid(Aβ)and intraneuronal tau containing neurofibrillary tangles(NFTs).By the age of 65,AD is divided into early onset AD(EOAD)and late onset AD(LOAD).Most of EGAD are caused by presenilin 1(PSEN1)gene,presenilin 2 gene and amyloid precursor protein(APP)gene mutation.The PSEN1 gene mutation is the most common one.When it occured,the protein activity decreased,resulting in the deposition of Aβ.More than 200 mutations have been found in PSEN1.Our team had reported an EOAD family with PSEN1 p.G378E mutation.The proband was 39 years old,and the pathological examination showed characteristic changes of EOAD:in the cerebral cortex,the neurons loss,astrocyte activation,Aβ42 deposition,and NFTs occured.Although the world’s research on AD has invested lots of financial and material resources,its pathogenesis is not completely clear and the treatment of AD is still a complex problem,which has led to the re-discussion of the pathogenesis of AD.Increasing evidence showed that chronic inflammatory responses played an important role in the pathology of AD,which directly or indirectly affects tau phosphorylation.In recent years,it has been found that the expression of tyrosine kinase binding protein(TYROBP)gene is up-regulated in brain of LOAD patients,and the TYROBP protein is mainly expressed in microglia in the brain.When it binds to the receptor,it can mediate inflammatory reactions and apoptosis through various signaling pathways.In the brain of AD transgenic mice,the mRNA of TYROBP was increased.What role does TYROBP protein play in AD pathology?Does TYROBP protein participate in the inflammatory response of brain tissue in AD patients caused by PSEN1 p.G378E,which affects the changes of AD pathology(tau phosphorylation)?These issues are worth to explore.In this study,we investigated the effect of TYROBP gene on phosphorylation of tau protein in PSEN1 p.G378E mice,and explored the effect of TYROBP protein on AD pathology,and to provide a idea for finding the new therapeutic target of AD.ObjectiveWe created AD mouse model with PSEN1 p.G378E gene mutation,and TYROBP knockout mice were also constructed.Wild type(WT),TYROBP knockout heterozygous(TYROBP-/+)and TYROBP knockout Homozygous(TYROBP-/-),PSEN1 p.G378E knock-in heterozygous(PSEN1G378E/WT)and PSEN1 p.G378E knock-in homozygous(PSEN1G378E/G378E),hybridization heterozygous(TYROBP-/+;PSEN1G378E/WT)and hybridization homozygous(TYROBP-/-;PSEN1G378E/G378E)mice were obtained.Behavioral changes,AD pathological changes,expression levels of inflammatory cells and inflammatory factors among different genotype mice were analyzed.The results could help us to investigate the effect of TYROBP gene on phosphorylation of tau protein in PSEN1 p.G378E mice,explore the role of TYROBP protein in the development of AD,understand the pathogenesis of AD,and provide a idea for finding the new therapeutic target of AD.Methods2-month-old TYROBP knockout heterozygous mice and PSEN1 p.G378E knock-in heterozygous AD mice were constructed by Cyngen Biosciences Inc.The heterozygous mice were purified and propagated,Wild type(WT),TYROBP knockout heterozygous(TYROBP-/+)and TYROBP knockout homozygous(TYROBP-/-),PSEN1 P.G378E knock-in heterozygous(PSEN1G378E/WT)and PSEN1 p.G378E knock-in homozygous(PSEN1G378E/G378E)mice were identified by DNA sequencing.TYROBP knockout homozygous(TYROBP-/-)and PSEN1 p.G378E knock-in homozygous(PSEN1G378E/G378E)mice were re-hybridized to obtain hybridization heterozygous(TYROBP-/+;PSEN1G378E/WT)and hybridization homozygous(TYROBP-/-;PSEN1G378E/G378E)mice.In this study,seven different genotype 5-month-old mice were used as the research objects.The behavior of different genotype mice was detected by water maze test.The swimming track of mice was recorded by ANY-maze software.Western blotting was applied to detect the phosphorylation level of tau protein and the expression of phosphorylation signaling pathway proteins in hippocampus among different genotype mice.The location of TYROBP expression and inflammatory cells(microglia and astrocyte)in mouse hippocampus were observed by immunofluorescence.The level of inflammatory factors including EL-6,IL-1β and TNF-α were detected by ELISA.SPSS 20.0 software was used for data analysis.Data are reported as mean ± SD.The mean of multiple samples was tested first for homogeneity of variance,and data was analyzed by one-way ANOVA.Otherwise,the Kruskal-Wallis H test was applied.Repeated-measures ANOVA is used in the water maze test.The difference was statistically significant at P<0.05.Results1.TYROBP knockout heterozygous mice and PSEN1 p.G378E knock-in heterozygous mice were successfully constructed in this study.Wild type(WT),TYROBP knockout heterozygous(TYROBP-/+)and TYROBP knockout Homozygous(TYROBP-/-),PSEN1 p.G378E knock-in heterozygous(PSEN1G378E/WT)and PSEN1 p.G378E knock-in homozygous(PSEN1G378F/G378E),hybridization heterozygous(TYROBP-/+;PSEN1G378E/WT)and hybridization homozygous(TYROBP-/-;PSEN1G378E/G378E)mice are obtained successfully.2.Results of water maze test:(1)In the positioning navigation training experiment,the escape latency of each genotype mouse decreased with the increase of training days(P<0.05),and the escape latency of PSEN1 p.G378E knock-in AD mice was higher than WT mice.On the fifth day,the escape latency of TYROBP knockout homozygous(TYROBP-/-)mice and hybridization mice(TYROBP-/-;PSEN1G378E/G378E)was shorter than that of PSEN1 p.G378E knock-in AD mice.(2)The search strategy of PSENl p.G378E knock-in AD mice was mainly "random",which consisted with the abnormal changes in swimming strategies of AD mice.Other mice mainly changed from "marginal" or "random" to "trend".(3)In the space exploration experiment,the number of accrossing platform and the target quadrant swimming time of PSEN1 p.G378E knock-in AD mice were significantly lower than those of WT mice(P<0.05),while there was no significant difference between WT mice and TYROBP knockout mice(P>0.05).3.Immunofluorescence results showed that TYROBP protein was mainly expressed in microglia.Western blot results showed that TYROBP protein increased in hippocampus of PSEN1 p.G378E knock-in AD mice,but the expression level of TYROBP was decreased in the hybridization mice and TYROBP knockout mice.4.The expression levels of total tau protein and phosphorylated tau protein:In TYROBP knockout mice,the expression levels of total tau protein,AT8 and T181 were higher than those in WT mice(P<0.05),and the expression of PHF1 and T231 protein were unchanged(P>0.05).In PSEN1 p.G378E knock-in AD mice,the expression levels of total tau protein,AT8,T181,T231,PHF1 protein were higher than those of other genotype mice(P<0.05).The levels of total tau protein and phosphorylated tau protein(AT8,T181 and T231)decreased in hybridization homozygous(TYROBP-/-;PSEN1G378E/G378E)mice compared with TYROBP knockout mice and PSEN1 p.G378E knock-in AD mice.5.The expression levels of phosphorylation signaling pathway proteins:(1)The levels of CDK5 and P-CDK5 proteins expressed:PSEN1 p.G378E knock-in AD mice and TYROBP knockout mice>hybridization homozygous mice>WT mice(P<0.01),but there was no significant difference between PSENI p.G378E knock-in AD mice and TYROBP knockout mice(P>0.05).(2)Expression levels of ERK and P-ERK proteins:The expression level of ERK protein in hybridization homozygous mice(TYROBP-/-;PSEN1G378E/G378E)was lower than that in other genotype mice(P<0.01).The expression level of P-ERK protein in TYROBP knockout mice was higher than that in WT mice,but there was no significant difference in the expression of ERK protein and P-ERK protein in other genotype mice(P>0.05).(3)Expression levels of GSK3P and P-GSK3β proteins:PSEN1 p.G378E knock-in AD mice>TYROBP knockout mice>hybridization mice>WT mice(P<0.01).6.Expression of inflammatory cells:TYROBP knockout mice and PSEN1 p.G378E knock-in AD mice>WT mice(P<0.05),and hybridization mice were lower than PSEN1 p.G378E knock-in AD mice.7.Expression of inflammatory factors:The expression of IL-6 in PSEN1 p.G378E knock-in AD mice was higher than that in other genotype mice(P<0.05);the expression of IL-1β in hippocampus of hybridization mice was lower than PSENl p.G378E knock-in AD homozygous type mice(PSEN1G378E/G378E)(P<0.05);the expression of TNF-α in hippocampus of each genotype mouse was not statistically significant(P>0.05).Conclusion1.TYROBP knockout heterozygous mice and PSEN1 p.G378E knock-in heterozygous mice were successfully constructed.Wild type(WT),TYROBP knockout heterozygous(TYROBP-/+)and TYROBP knockout Homozygous(TYROBP-/-),PSEN1 p.G378E knock-in heterozygous(PSEN1G378E/WT)and PSEN1 p.G378E knock-in homozygous(PSEN1G378E/G378E),hybridization heterozygous(TYROBP-/+;PSENIG378E/WT)and hybridization homozygous(TYROBP-/-;PSEN1G378E/G378E)mice are obtained successfully.2.The mice with PSEN1 p.G378E mutation is successful AD mouse model.3.TYROBP protein is expressed increasedly in the hippocampus of PSEN1 p.G378E knock-in AD mice,which is mainly expressed in microglia.4.In AD mouse model with PSEN1 p.G378E mutation,tau phosphorylation(AT8,T181,T231,PHF1)is mainly mediated by GSK-3β and CDK5 signaling pathways,and TYROBP gene deficiency could decrease the phosphorylation level of tau protein in AD.5.Inflammatory response is involved in the pathological changes of AD,TYROBP gene deficiency could improve the situation by reducing the number of microglia and astrocytes,reducing the expression of inflammatory factor IL-6.6.TYROBP gene deficiency could improve the spatial learning ability of AD mice to some extent,but have no significant improvement on spatial memory ability.