| BackgroundAlzheimer's disease(AD)is a slow-onset,progressive neurodegenerative disease.With the acceleration of population aging,the incidence of AD has increased sharply and become a serious threat to the physical and mental health of the elderly.Early studies mainly considered that amyloid plaques formed by A? and neurofibrillary tangles formed by Tau are the two main causes of AD,and developed a variety of drugs targeting these two targets.Although encouraging results have been show n in animals,few drugs have been show n to be effective in clinical trials.Therefore,it is extremely urgent to find other potential pathogenic factors of AD and develop corresponding drugs.In recent years,several studies have found that the loss and dysfunction of neuronal synapse appear in the early stage of AD,and there is a more significant correlation between this feature and cognitive impairment in AD.Micro RNAs,as small-molecule RNAs of post-transcriptional regulation,are widely involved in various physiological processes,including development,learning and memory,tumorigenesis and so on.A variety of miRNAs have been found to be involved in the occurrence of neurological diseases such as Parkinson's disease(PD)and AD.In the early stage of our research group,we also found the role of miR-124,miR-101 b and miR-218 in Tau lesions,synaptic injury and learning and memory impairment.However,the role and mechanism of miRNA related to neuronal synaptic activity in learning and memory and AD are not very clear.ObjectiveTo investigate the changes of miRNAs related to synaptic activity of neurons and to clarify their role and potential mechanism in AD.MethodsThe synaptic activity-related miRNAs enriched in the hippocampus of mice were screened by means of literature retrieval and functional analysis.RT-q PCR was used to detect the changes of these miRNAs in the hippocampus of 12-month-old and 9-month-old APP/PS1 and the age-matched control C57 mice,and the miRNA with significant change was selected for follow-up verification.The changes of miRNA were further verified in APP/PS1 mice of different months.RNA fluorescence in situ hybridization(FISH)technology was used to analyze the location of miRNA.The transcriptional production and degradation level of miRNA were evaluated by RNA stability assay and luciferase reporting experiments,and the cause of miRNA change was determined.Overexpression or inhibition of miRNA expression was achieved by stereotactic injection of the hippocampus,followed by Morris water maze,electrophysiology,and Golgi staining to determine the role and mechanism of miRNA in AD.ResultsWe found that miR-135 a decreased most significant by RT-q PCR among those miRNAs associated neuronal activity.Through the RNA stability assay,we found the degradation of miR-135 a didn't increase.Instead,through analysing the transcription of miR-135 a,we found the transcription down-regulation of pri-miR-135a-1.Further through bioinformatics analysis and chromatin immunoprecipitation(Ch IP)experiment,we have shown that the transcription factor Foxd3 can bind to and activate the promoter of pri-miR-135a-1.At the same time,we found that the protein content of Foxd3 in the hippocampus of APP/PS1 mice was also down-regulated.Artificial down-regulation of miR-135 a in the hippocampus of C57 mice can induce synaptic injury and learning and memory impairment of hippocampal neurons.Conversely,restoring the expression of miR-135 a can effectively improve synaptic loss and learning and memory impairment of AD in APP/PS1 mice.Conclusions1.Among the many miRNAs related to neuronal activity in the hippocampus of AD mice,the down-regulation of miR-135 a is the most significant.2.In AD,the down-regulation of Foxd3 leads to the transcriptional inhibition of pri-miR-135a-1,thus reducing the expression of miR-135 a.3.In AD,the decrease of miR-135 a can lead to synaptic loss,neuron morphological abnormality,LTP damage and learning and memory impairment.Overexpression of miR-135 a can reverse the above phenotype.Background In the first part of the study,we found that the expression of miR-135 a began to be down-regulated in the early stage of AD mice,and this down-regulation had no effect on the presynaptic function of hippocampal neurons in AD mice,but mediate synaptic disorders and learning and memory impairment via interfering the morphology and function of postsynaptic dendritic spines in AD.However,it is not clear which molecule miR-135 a targets to regulate the morphology and function of neuronal dendrites and dendritic spines.Cytoskeleton is an important structure to maintain cell morphology and regulate intracellular material transport and distribution,especially in cells with complex morphology such as neurons.Actin cytoskeleton in neurons is widely involved in important biological processes such as neuronal morphogenesis,axonal growth,dendritic branching and stabilization,development of dendritic spines and synaptic plasticity.The changes of actin skeleton have been observed in various neurodegenerative diseases,such as AD,Parkinson's disease(PD),Huntington's disease(HD)and so on.Targeting the actin cytoskeleton of neurons may provide a new direction for synaptic loss and plasticity changes in early stage of AD.Dendritic spines are the key parts of synaptic information transmission in mammals.The morphological changes,maintenance and regulation of dendritic spines are very important for synaptic plasticity and learning and memory.All kinds of postsynaptic actin binding proteins and kinases can participate in the regulation of synaptic structure and functional plasticity by regulating actin skeleton.Therefore,it is necessary to study w hether the target gene of miR-135 a is involved in the regulation of synaptic disorders and learning and memory disorders in AD.Objective To investigate whether the target gene of miR-135 a is involved in the regulation of actin skeleton,and then regulate the learning and memory impairment in AD and its molecular mechanism.Methods The target molecules involved in actin cytoskeleton regulation of miR-135 a were identified by bioinformatics prediction,biochemical analysis and luciferase reporting experiments.The substrates regulating actin of Rock2 were screened by literature search and bioinformatics prediction,and the phosphorylation changes of the substrates were verified by transfection of miR-135 a mimics or inhibitors to artificially regulate the expression of Rock2.Morris water maze,electrophysiology and Golgi staining were used to determine whether silent Rock2 could reverse synaptic loss and learning and memory impairment in AD.A polypeptide fused with TAT(Human Immunodeficiency Virus type 1 transcriptional activator)was designed to block the phosphorylation of Add1 Ser726 site and to verify whether it can improve learning and memory impairment of AD in APP/PS1 mice.Results The target genes of miR-135 a were predicted by Targetscan and miRDB.The KEGG pathway enrichment analysis showed that these genes were mainly involved in regulation of actin skeleton and calcium signal.Through biochemistry experiment,it is proved that Rock2 is the target gene dow nstream of miR-135 a.And Rock2 can promote the phosphorylation of adducin(Add1)at Ser726 site.Through stereotactic injection of sh-Rock2 virus into hippocampus,we proved that silencing Rock2 can restore learning and memory impairment in AD.Finally,we designed a TAT fusion peptide si P-Add1 and demonstrated that it can inhibit the phosphorylation of Add1 Ser726 site in cells and animals.It can also improve the synaptic loss,dendritic branches and learning and memory impairment of hippocampal neurons in APP/PS1 mice.Conclusions 1.The down-regulation of miR-135 a in the hippocampus of APP/PS1 mice can lead to an increase of Rock2 protein expression.2.The high expression of Rock2 induces increased phosphorylation of Add1 Ser726 site,which leads to the instability of actin skeleton,and then leads to synaptic loss and learning and memory impairment.3.Silencing Rock2 or using si P-Add1 to interfere with Add1 phosphorylation can improve the above phenotype. |
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