MicroRNA-137 Inhibits Tau Hyperphosphorylation By Targeting CACNA1C In Alzheimer’s Disease

Objective:Alzheimer’s disease(AD)is a neurodegenerative disease,characterized by memory loss,multiple cognitive impairments,and intellectual impairments.Currently,there are 5.4 million Americans living with AD,and this number is estimated to increase up to 16 million by 2050.In the worldwide,AD will affect more than 35 million persons by 2050.This disease contributes to significant social and economic burdens worldwide.Although extensive researches have been dedicated to the identification of AD process,the AD pathogenesis is not completely known.To date,research into AD reveal that AD begins in learning and memory regions of the brain,then spreads to hippocampus,temporal cortex,frontoparietal cortex,and to subcortical nuclei.Accumulated evidences show that AD is associated with multiple cellular changes,including synaptic loss,mitochondrial dysfunction,neuronal loss,oxidative damage,amyloid beta(Aβ)formation and accumulation,phosphorylated tau(p-tau)formation and accumulation,and inflammatory responses.However,no early detectable biomarkers for AD were found,and no drugs can delay or prevent AD progression.Most of the research into AD illustrate that microRNAs(miRNAs)are involved in above cellular changes.MiRNAs are short,single-stranded,non-coding RNAs that play crucial roles in the posttranscriptional regulation of gene expressions.MiRNAs are emerging as key players in multiple human diseases,including cardiovascular diseases,nephropathy,cancer,and several neurodegenerative diseases such as schizophrenia,Parkinson’s,and AD.Previous studies demonstrate that miRNAs play crucial roles in neurogenesis,neuronal maturation and brain development.Among the miRNAs,several miRNAs have been identified in AD patients that regulate the key AD genes,such as:microRNA(mi R)-101 down-regulates the amyloid precursor protein(APP)and fibrillar Aβin hippocampal neurons,miR-219 and miR-34 down-regulate tau protein,miR-26b up-regulates Retinoblastoma 1(Rb1,critical component of cell cycle and apoptosis pathways),miR-30a-5p and miR-206 down-regulate brain-derived neurotrophic factor gene(BDNF).All the results demonstrate that dysfunction of mi RNA signaling results neurodegenerative and psychiatric disorders.However,miRNA that may be responsible for AD,are not completely known and should be further investigated.Human miR-137(hsa-miR-137)is one miRNA of particular interest in the field of pathergasiology.MiR-137,one brain-enriched miRNA,has been identified as being responsible for neurodegenerative diseases such as schizophrenia,and AD.The largest genome-wide association study(GWAS)for schizophrenia(SCZ)determines rs1625579 as the strongest new association with SCZ,located in an intron of a primary transcript for mi R-137.Association analyses illustrate that miR-137 and its target gene CACNA1C(α-1C subunit of the L-type voltage-gated calcium channel gene)are strongly associated with both SCZ and bipolar disorder(BP).However,data about the roles of miR-137 in the pathogenesis of AD are highly limited,which are only reported by Geekiyanage et al.Hence,in this study,we investigate that how mi R-137 regulates AD in APP/PS1transgenic mice and in human neuronal SH-SY5Y cells.In vivo studies,we assess the expression levels of miR-137 and its target gene CACNA1C in hippocampus and cerebral cortex of AD mice,and also evaluate the expression levels of Aβ1-40 and Aβ1-42 in serum,hippocampus and cerebral cortex of AD mice.In vitro studies,regulation effects of miR-137 on activity of wild-type(WT)or mutant(Mut)CACNA1C,and expression levels of CACNA1C and p-tau(Ser202,Ser396,and Ser404 sites)are investigated.This study will explain how miR-137 regulates the process of AD,and provide a novel therapeutic target for AD.Methods:1.Animals and Antibodies:The C57BL/6 mice and B6C3-Tg(APPswe,PSEN1dE9)85Dbo/J mice(AD)were purchased from Nanjing Biomedical Research Institute of Nanjing University(Nanjing,China).Primary antibodies for CACNA1C(1:200;Santa cruz,Santa Cruz,CA,USA),p-tau(Ser 202)(1:400;Boster,Wuhan,China),p-tau(Ser 396)(1:5000;Abcam,MA,USA),p-tau(Ser 404)(1:500;Sangon,Shanghai,China),tau(1:500;Sangon)and Aβ(1:100;Sigma,St.Louis,USA)were used in this study.In our study,the animals were maintained in accordance with the Institutional Animal Care and use Committee and study protocol was approved by the local ethics committees.2.Morris water maze(MWM):A MWM(120 cm in diameter,40 cm in height with a water depth of 24 cm)(Anhui zhenghua biological instrument equipment co.,LTD,Anhui,China)was performed to detect spatial learning and memory ability of the mice.The MWM apparatus was split into four quadrants and a platform(9cm in diameter,23 cm in height)was placed in the third quadrant.The MWM test consisted of two parts:a place navigation test from day1 to day5 and a spatial probe test on day6.During the place navigation text,four contiguous trails were performed each day.Each mouse was allowed an adaptation period of 20 s on the platform,then placed in each quadrant respectively,and given 60 s to reach the platform.The mice reached the platform within 60 s were remained on the platform for 5 s,while the mice were manually guided to the platform if the mice could not reach the platform within 60 s and remained on the platform for 10 s.During the spatial probe test,the platform was removed and the mice were placed into the first quadrant.The escape latency,swimming path,and target zone frequency of the mice were recorded.3.Total RNA extract,cDNA synthesis and Real-time PCR(RT-PCR):Total RNA was isolated by using RNApure total RNA fast isolation kit according to the manufacturer’s instructions(BioTeke,Beijing,China).cDNA was synthesized by using Super M-MLV reverse transcriptase according to the manufacturer’s protocol(BioTeke).Two pair primers(miR-137-F/R and U6-F/R)were designed for expression analysis(Table 1).Real-time PCR was performed with SYBR GREEN master mix(Solarbio,Beijing,China)on Exicycler 96(Bioneer,Daejeon,Korea).Data was determined via the 2-ΔΔCT method.4.Western blot:The tissues and cell samples were collected and lysed with RIPA buffer(Beyotime,Beijing,China)containing 1%phenylmethylsulphonyl fluoride(PMSF,Beyotime).The protein concentration was measured by BCA Protein Assay Kit(Beyotime).Equal amounts of protein samples were separated on a 10%SDS-PAGE and transferred onto a PVDF membrane(Millipore,Bedford,MA,USA).The membrane was blocked with 5%skim milk and incubated with primary antibodies and HRP-labeled Goat Anti-Rabbit IgG(H+L)secondary antibody(1:5000;Beyotime).Proteins were visualized by ECL Reagent(7sea biotech,Shanghai,China)and imaged with a gel imaging system(Liuyi,Beijing,China).β-actin was used as an internal control.5.Enzyme-linked immunosorbent assay(Elisa):The levels of Aβ1-40 and Aβ1-42 were measured by Elisa assay using Elisa Kit for Aβ1-40 and Aβ1-42(USCN,Wuhan,China)following the manufacturer’s instructions.The values at 450 nm were read using a microplate reader(BioTeke,Winooski,VT,USA).6.Immunofluorescence(IF)assay:The paraffin-embedded 5-μm cerebral cortex sections and hippocampal sections were dewaxed with xylene and rehydrated with alcohol.After that,the sections were subjected to microwave antigen retrieval for 10 min and blocked with goat serum(Zsgb-bio,Beijing,China).The sections were co-incubated with primary antibodies for CACNA1C and Aβovernight at 4°C,then co-incubated with FITC-labeled Goat Anti-Mouse IgG(H+L)and Cy3-labeled Goat Anti-Rabbit IgG(H+L)secondary antibodies.The nucleus was staining with DAPI(Beyotime).7.Cell culture,plasmid construction and transfection:SH-SY5Y cells were cultured in Dulbecco’s modified Eagle’s medium(DMEM;Gibco,MA,USA)containing 10%fetal bovine serum(Hyclone Co.,Logan,USA)in 37°C in a humidified atmosphere of 5%CO2.The wild-type CACNA1C and mut-CACNA1C 3’-UTR were inserted into the vector pmirGLO(Promega,WI,USA).The cells(4×10~5)were transfected with miR-137mimics or miR-137 inhibitor,and corresponding NC miRNAs for the later experiment.Forty-eight hours after the transfection,partial transfected cells were treated with Aβ1-42(5μM;GL Biochem,Shanghai,China)for 24h.Then the cells were collected for the later experiments.8.Luciferase assay:SH-SY5Y cells(1×10~5)were cotransfected with pmirGLO-CACNA1C or pmirGLO-mut-CACNA1C 3’-UTR luciferase reporter plasmids,and either miR-137 mimics or NC miRNA,using a Lipofectamine 2000 reagent(Invitrogen,Carlsbad,CA,USA).The luciferase assay was performed with Dual-Luciferase~?Reporter Assay System according with the manufacturer’s instructions(Promega).9.Statistical analysis:Statistical analysis was performed using Graphpad 6.0.Differences between two groups were determined using Student’s t-test.All experiments were replicated at least 3 times and data was presented with standard deviation.P<0.05was considered as significant.Results:1.Behavioral tests:A MWM text was used to assess the spatial learning and memory ability of the control and APP/PS1 mice.The escape latency to reach the platform in AD APP/PS1 mice was significant longer than that in healthy control mice at day 4 and day 5.Results from the spatial probe test showed that the number of crossing the platform and the percentage of time in the target quadrant in APP/PS1 group was significantly decreased as compared to the control group.These data indicated that spatial learning and memory ability of APP/PS1 mice declined significantly.2.Expression levels of miR-137,Aβ1-40 and Aβ1-42 in APP/PS1 mice:Results from RT-PCR showed that the expression levels of miR-137 were significantly lower in hippocampus and cerebral cortex of APP/PS1 mice as compared to control mice.APP/PS1 mice acquired increased levels of Aβ1-40 and Aβ1-42 in serum,hippocampus and cerebral cortex.Further,IF assay showed that APP/PS1 mice acquired a higher level of CACNA1C and Aβplaques in both hippocampus and cerebral cortex.3.Effects of miR-137 on CACNA1C activity and expression in SH-SY5Y cells:In SH-SY5Y cells,miR-137 inhibited the activity of WT-CACNA1C,but not modulated the activity of mut-CACNA1C.Further,miRNA-137mimics decreased the protein expression level of CACNA1C,and miR-137 inhibitor increased CACNA1C level in SH-SY5Y cells.4.Effects of mi R-137 on Aβ1-42 induced expression levels of p-tau(Ser 202),p-tau(Ser 396),p-tau(Ser 404)and tau.We further examined whether miR-137 mimics and miR-137 inhibitor modulated the expression levels of phosphorylated and non-phosphorylated tau.The results indicated that Aβ1-42significantly up-regulated the expression levels of p-tau(Ser 202),p-tau(Ser 396)and p-tau(Ser 404)in SH-SY5Y cells.MiR-137 mimics markedly inhibited Aβ1-42 induced p-tau expressions,and miR-137 inhibitor increased Aβ1-42 induced p-tau expressions.MiR-137 mimics and miR-137 inhibitor had no clear effect on the expression level of non-phosphorylated tau.Conclusions:The findings of this study,using a double-transgenic mouse model of Alzheimer’s disease(AD)and human neuroblastoma SH-SY5Y cells,have shown that expression levels of micro-RNA-137(miR-137)were low in AD mice,which might indicate the regulatory function of mir-137 in the pathogenesis of AD by inhibiting the hyperphosphorylation of the tau protein.Also,miR-137 regulated the expression of the calcium voltage-gated channel subunit alpha-1 C(CACNA1C)gene by directly binding to the 3’-UTR of CACNA1C in SH-SY5Y cells.The findings of this study have shown a novel interaction between miR-137 and CACNA1C,which suggests that miR-137 may be a potential diagnostic or therapeutic target for AD.