Down-regulation Of MiR-369 Deteriorates Cognitive Impairments And Increases The Phosphorylation Of Tau Protein In 3xTg-AD Mice

There are estimated 6 million people who are living with Alzheimer’s disease(AD)in China and about 300,000 new cases annually.However,the pathogenesis of AD is complex and remains unclear,and the therapy is limited.Based on known knowledge,the two major pathologic hallmarks of AD are the presence of amyloid-beta plaques and neurofibrillary tangles induced by hyperphosphorylated tau protein,but the mechanisms involved in their formation are not well understood.Micro-RNAs(miRNAs)are a class of small non-coding RNAs and are involved in the regulation of gene expression.Recent studies have revealed that there are many specific changes in miRNAs in the brain of AD patients and these changes play an important role in AD pathogenesis.In the preliminary research of this study,we searched the dataset GSE16759 of Gene Expression Omnibus(GEO)database regarding to expression of miRNAs in AD patients,and found that miRNAs-369(miR-369)expression is significantly decreased in AD patients.Furthermore,miR-369 is a conserved miRNA with 100% sequence identity between human and mouse.Therefore,in this study we investigated the effect of miR-369 downregulation on cognitive behaviors of 3xTg-AD model mice and the mechanism that miR-369 regulates the phosphorylation level of tau protein via target genes.Part Ⅰ The expression of miR-369 in brain of 3xTg-AD mice and its role in cognitive behavioral changesObject: To confirm the down-regulation of miR-369 expression according to the preliminary search on the dataset GSE16759,and then provide foundations for further research on miR-369 in AD pathogenesis.Method: The expression of miR-369 in the cerebral cortex in 3xTg-AD and wild type C57 mice was investigated using real-time q PCR technique.The cognitive behaviors of the mice were investigated using Morris water maze and Barnes maze test.Result: In 3xTg-AD mice,the expression level of miR-369 in the cerebral cortex decreased significantly in 6-month-old age compared to2-month-old age,whereas in wild type C57 mice there is no significant difference in miR-369 expression between 6-month-old and 2-month-old ages.Simultaneously,the miR-369 expression level in the cerebral cortex of6-month old 3xTg-AD mice is significantly lower compared to 6-month-old wild type C57 mice.In addition,the cognitive behaviors of 3xTg-AD mice were clearly impaired at the time point of 6-month-old age compared to the wild type C57 mice.Conclusion: The miR-369 level in the cerebral cortex significantly downregulated with the increase of age in 3xTg-AD mice,and the cognitive ability deteriorates accompanied with the downregulation of miR-369 level.Part Ⅱ miR-369 regulates level of phosphorylated tau protein via targeting Fyn and SRPK2Object: To investigate whether miR-369 can bind 3’-UTR of Fyn and SRPK2,and then decrease the expression of Fyn,SRPK2 and the level of phosphorylated tau protein.Method: Through the website of target scan,potential targets of miR-369 were searched.Using luciferase reporter assay,the 3’-untranslated region(UTR)of potential targets binding with luciferase reporter gene was co-transfected into 293 T cells with miR-369,and then luciferase activity was measured using Luciferase Assay System.Using cultured mouse neuroblastoma cell line neuro-2a,the expressions of miR-369 and its potential target genes and proteins were upregulated or knockdown by transfection of its plasmid or its si RNA,respectively,and then the expression levels of miR-369,Fyn or SRPK2 and phosphorylated tau protein were investigated using real-time q PCR and Western blot analysis.Result: The target website search analysis showed that miR-369 can bind3’-UTR of Fyn or SRPK2,which suggested that miR-369 could target both Fyn and SRPK2 kinases.The luciferase assay results showed that co-transfection of miR-369 markedly reduced luciferase activity of FYN or SRPK2 reporter construct individually.The transfection of miR-369 in neuro-2a cells significantly upregulated the expression of miR-369,which in turn downregulated the expressions of Fyn and SRPK2 in m RNA and protein levels and decreased the level of phosphorylation tau protein.In contrast,transfection of miR-369 si RNA significantly increased the level of phosphorylation of tau protein.In addition,it was shown that transfection of si RNA against Fyn or SRPK2 significantly decreased the level of phosphorylation of tau protein.Conclusion: miR-369 can target 3’-UTR of Fyn or SRPK2 and then decrease their expression level,which would inhibit the phosphorylation of tau protein.Part Ⅲ The effect of miR-369 loss on cognitive ability and the level of phosphorylated tau protein in the brain in 3xTg-AD miceObject: To investigate the effect of miR-369 loss in the brain the level of phosphorylated tau protein,and investigate whether miR-369 loss can affect cognitive ability in the brain in 3xTg-AD mice,and then to elucidate the mechanism of loss of miR-369 in the pathogenesis of AD.Method: Using gene knockout and hybridization techniques,we knockout the miR-369 of 3xTg-AD mice.Besides,the restore of the miR-369 level in the brain of miR-369 knockout 3xTg-AD mice was applied by intracerebroventricular injection.Thereafter,the cognitive function of the mice was evaluated by Morris water maze and Barnes maze test.The expressions of miR-369,Fyn,SRPK2,tau protein and phosphorylated tau protein in the cerebral cortex were examined by realtime q PCR,Western blot or immunochemistry.Result: Morris water maze and Barnes maze tests showed that miR-369 loss deteriorated the cognitive behaviors and memory ability in 3xTg-AD mice.Real-time q PCR,Western blot and immuno-histochemistry assays showed that the expression of Fyn and SRPK2 kinases and the phosphorylated tau protein increased significantly in the miR-369 knockout mice.In contrast,restore of miR-369 in the miR-369 knockout mice significantly decreased the expression of Fyn and SRPK2 kinases and phosphorylation level of tau protein.Conclusion: Loss of miR-369 promotes phosphorylation level of tau by upregulating the expression of Fyn and SRPK2 kanases,which contributes AD pathogenesis and deteriorates cognitive ability of AD mice.