| BackgroundAlzheimer's disease(AD)is a rapidly progressive neurodegenerative disease.According to the report of the World Health Organization,there are about 50 million people suffering from dementia in the world,among which Alzheimer's disease is the most common type.The main clinical features of AD are extracellular ?-amyloid deposition and neurofibrillary tangles formed by intracellular hyperphosphorylation of Tau.In addition,AD is characterized by progressive neuron loss,synaptic dysfunction,abnormal mitochondrial function,inflammatory response and so on.Although many advances have been made in the pathogenesis of AD and the regulation of synaptic plasticity,there is still no drug that cures effectively AD,and it is still necessary to study the molecular mechanism of AD in detail.Micro RNA(mi RNA)is a small RNA molecule with about 22?25 nucleotides,which is widely expressed in humans,plants,fungi,bacteria and viruses,and has good conservation among species.Mi RNA mainly suppresses gene expression by binding to the 3'-untranslated region(3'-UTR)of m RNA in an incompletely complementary pairing manner,thus participating in the process of individual development and disease occurrence.Recently,there is some evidence that mi RNA is involved in the regulation of neurodegenerative diseases.However,the study of mi RNA in regulation of A?,p-tau and synaptic plasticity needs further exploration.It is urgent to investigate mi RNA function in AD and this will provide valuable targets for new therapies to prevent or delay AD symptoms.In recent years,adenosine and its receptor have attracted much attention because of its role in normal physiological conditions and many neurological diseases.There are four G protein-coupled adenosine receptors(A1,A2 A,A2B and A3),which are widely distributed in the brain.Adenosine regulates neuronal activity and affects many brain functions such as cognition and memory,neuronal injury and degeneration.Adenosine receptor A1(A1R)is an inhibitory adenosine receptor which is highly expressed in brain,especially in hippocampus.Although in some early studies,it was found that A1 R decreased in AD brain by using autoradiography receptor binding method,recent studies showed that the expression of A1 R increased significantly in neurons with AD-specific pathological changes.In addition,the up-regulation of A1 R was found in the 5x FAD mice with fear memory impairment.Activation of A1 R triggers neuronal death and A1 R antagonist alleviates axonal lesion in human Tau transgenic mice with the ?K280 mutation,suggesting that A1 R antagonist plays a key role in mediating AD pathology and cognitive function.However,it is still unclear how A1 R activation leads to cognitive and synaptic damage in Alzheimer's disease.ObjectiveTo explore the role and molecular mechanism of adenosine receptor A1 in Alzheimer's disease.MethodsThe expression of A1 R was detected by Q-PCR,western blot,immunohistochemistry and immunofluorescence,and the potential mi RNAs regulating A1 R were identified by target gene prediction software.The expression of mi R-133a-3p in AD was detected by Q-PCR and FISH.Transcription factors regulating mi R-133a-3p were determined by transcription factor prediction software,and their regulatory relationships were determined by promoter luciferase in vitro.The regulation of mi R-133a-3p on A1 R was verified by using agomir and antagomir of mi RNA in vitro.To investigate the function of mi R-133a-3p and A1 R,mice were injected with mi R-133a-3p agomir or antagomir,adeno-associated virus or lentivirus of A1 R,and transgenic knockout technology.Also,the learning and memory of mice were evaluated by Morris water maze and Y-maze.The synaptic plasticity of neurons was evaluated by Golgi staining and electrophysiological LTP.RNA sequencing technology was used to identify the downstream gene expression after A1 R knockout and the expression of Lcn2 in AD was detected.Then,the function of Lcn2 on learning/memory and synaptic plasticity were investigated by using primary neurons in vitro or mice in vivo.ResultsWe found that A1 R was abnormally increased in the brains of AD mice and AD patients by WB,immunofluorescence and immunohistochemistry,but the m RNA levels of A1 R remained unchanged by Q-PCR.The results of mi RNA target prediction and Q-PCR indicated the candidate mi R-133a-3p.The mi R-133a-3p agomir inhibited,whereas the mi R-133a-3p antagomir promoted,the expression of A1 R by Q-PCR and western blotting,which further confirmed by dual luciferase assay in vitro.Additionally,the expression of mi R-133a-3p in AD was significantly decreased.The analysis of promoter prediction showed that the expression of mi R-133a-3p may be regulated by transcription factor Mef2 c,which was confirmed by the promoter of dual luciferase assay.Subsequently,interference with mi R-133a-3p or overexpression of A1 R in C57 mice led to learning and memory impairments in vivo.Overexpression of mi R-133a-3p or interference with A1 R in3×Tg-AD mice rescued the learning/memory impairments and synaptic injury.Furthermore,genetic knockout of A1 R also prevented the learning and memory impairment and synaptic injury in the 3×Tg-AD mice.Finally,the expression of Lcn2 was inhibited in the A1 R knockout mice and significantly increased in 3×Tg-AD mice.After interfering with the expression of Lcn2 in 3×Tg-AD,it ameliorated the learning and memory impairment and synaptic damage.ConclusionThe abnormal increment of A1 R in AD is regulated by mi R-133a-3p at transcription level.The expression of mi R-133a-3p also decreased due to the downregulation of transcription factor Mef2 c in AD,which further resulted in abnormal protein levels of A1 R.The inhibition of mi R-133a-3p or the overexpression of A1 R led to learning and memory impairment in wild-type mice,while the overexpression of mi R-133a-3p or the interference with A1 R in AD alleviated memory impairment and synaptic injury.Finally,A1 R knockout protected AD from learning and memory impairment and synaptic dysfunction through downstream Lcn2. |
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