Role And Mechanisms Of Hippocampus Long Non-coding RNA AK090335 On Learning And Memory Of Alzheimer's Disease Model Mice By Regulating Transthyretin

Background: Alzheimer's disease(AD)is an irreversible neurodegenerative disease,characterized by memory deficiency and cognitive impairment.Recent years,a large number of coding RNAs have being found their antisense transcripts,most of which are non-coding RNAs.Long non-coding RNAs(lnc RNAs)play an important role in the development and degradation of nerve system,as well as neurodegenerative diseases,by regulating the transcription of their target genes.The level of transthyretin(TTR)decreases with age.Besides,the latency of 5-month TTR knockout mice to find platform in Morris water maze was prolonged,as well as the 7-month TTR knockout mice showed significantly impairment of learning and memory.But the mechanism of TTR-induced learning and memory impairment was unclear.We have carried out this research in order to find out whether there exists any non-coding RNA to specifically regulate TTR.By consulting messages in databases,we identified a lnc RNA that shows reverse complement characteristics with TTR,and further explored the regulatory mode of lnc RNA AK090335 on TTR,as well as the key role of this regulatory mode in Alzheimer's disease.Objective: To explore the regulation mode of lnc RNA AK090335 on TTR and whether the regulation improves the spatial learning and memory of AD mice.Methods: APPswe/PS?E9 double transgenic AD mice were used in this research.Firstly,the RNA and protein levels of TTR in hippocampus of 6 months,9 months and 12 months AD mice were detected by real-time quantitative fluorescence PCR(RT-q PCR)and Western Blot.The reverse complement transcript AK090335 was found by consulting UCSC database,and the level of AK090335 in hippocampus of AD mice at different ages was detected.In order to further determine the characteristics of lnc RNA AK090335,specific primers were designed for RACE(rapid-amplification of DNA ends)experiments after consulting the reference sequence of AK090335 in NCBI database.The full-length sequence of lnc RNA AK090335 was constructed by comparing the reference sequence with purifying and sequencing the nested PCR products.Using molecular cloning technology,lnc RNA AK090335 was constructed on plasmid vector pc DNA4.0.Then,pc DNA4.0-AK090335 was transfected into neuroblastoma(N2a)cells to overexpress AK090335.RNA and protein were extracted to detect the expression of TTR.AAV-AK090335 virus were overexpressed in hippocampus DG region of 6-month-old male APP/PS1 mice by stereotaxic injection technique.One month later,learning and memory ability of these mice was tested by Morris water maze and fear conditioning,and the level of TTR in hippocampus were detected to determine whether the behavioral changes of mice were caused by the regulation of TTR by AK090335.Results: The expression of TTR in AD model mice decreased with the increasing of age,and the expression of lnc RNA AK090335 also showed the same downward trend,suggesting that lnc RNA AK090335 may lead to the occurrence and development of AD by regulating the expression of TTR.Results of Race showed that 5' terminal sequence of AK090335 matched the reference sequence perfectly,but the 3' terminal sequence did not match the reference sequence completely,so we renewed a new full-length sequence of lnc RNA AK090335.Overexpression plasmid pc DNA4.0-AK090335 was transfected into N2 a cells and the expression of AK090335 was increased in N2 a cells.The results of RT-q RCR and Western Blot showed that the RNA and protein levels of TTR increased with the increase of AK090335 expression,suggesting that AK090335 could positively regulate the expression of TTR in neuron.The expression of AK090335 was increased by injecting AAV-AK090335 virus into DG of 6-month-old APP/PS1 mice.The learning and memory ability was observed by water maze and fear conditioning.In Morris water maze,the latency of mice injected with AAV-AK090335 to find the platform was significantly shortened in the training stage,and the time spent in the target quadrant as well as times crossing the platform was significantly increased in the spatial exploration stage,suggesting that overexpression of AK090335 significantly improved learning and memory.In fear conditioning test,freezing percentage of mice overexpressing AK090335 increased significantly in the same content,indicating that fear memory was enhanced.RT-q PCR and Western Blot showed that the RNA and protein levels of TTR in hippocampus of mice overexpressing AK090335 were increased,suggesting that AK090335 could improve impaired spatial learning and memory ability of AD mice by increasing the expression of endogenous TTR.Conclusion: The expression of TTR and its antisense lnc RNA AK090335 in the hippocampus of AD mice decreases with age.Downregulation of TTR and AK090335 in hippocampus resulted in learning and memory impairment similar to APP/PS1 mice.Renewed AK090335 sequence was offered according to reference databases and experimental results.Upregulation of AK090335 in the hippocampus of AD mice could improve learning and memory impairment related to hippocampus,and this improvement was achieved by increasing the expression of endogenous TTR.Therefore,we could conclude that AK090335 played an important role in improving learning and memory impairment of AD by increasing TTR level.