| Alzheimer’s disease（AD）is a neurodegenerative disease with progressive cognitive decline and learning and memory impairment as the main clinical manifestations,but its pathogenesis is still unclear.In recent years,circular RNA（circ RNA）has been widely recognized and valued in many biological fields.The expression of circ RNA is abundant in the nervous system,and it can participate in multiple physiological and pathological processes such as synaptic plasticity,learning and memory through epigenetic,transcriptional and post-transcriptional regulation.Dube U et al.[1]analyzed the differential expression of circ RNA in brain tissues of AD patients and found that circ-Bptf was significantly correlated with the diagnosis of AD,severity of clinical dementia and severity of neuropathology,suggesting that circ-Bptf plays an important role in the pathogenesis of AD.However,the specific mechanism of its action still needs further study.Objective:By studying the expression of circ-Bptf in APP/PS1 mice with learning and memory disorders and its molecular regulatory mechanism,this study clarified the function of circ-Bptf and provided new ideas and insights for improving AD-related learning and memory disorders.Methods:1.Animal level1.1 In the hippocampus of APP/PS1 mice,the expression of circ-Bptf was detected.APP/PS1 mice at 3,6,9,and 12 months of age and corresponding control WT mice were used in the experiment.Four mice at 3,6,and 9months of age were randomly selected,and 14 mice at 12 months of age were randomly divided into wild group（WT）and APP/PS1 group（APP/PS1）.The changes of learning and memory ability in APP/PS1 mice were detected.q RT-PCR was used to detect the expression of circ-Bptf in the hippocampus of APP/PS1 mice.1.2 Effects of hippocampal overexpression of AAV-circ-Bptf on learning and memory ability in APP/PS1 mice.GFP adeno-associated virus control vector（AAV-GFP）and circ-Bptf overexpression adeno-associated virus vector（AAV-circ-Bptf）were injected into the CA1 region of mouse hippocampus using hippocampal stereotaxic technique.Twelteen-month-old APP/PS1 mice and corresponding control WT mice were divided into three groups:WT mice+GFP adeno-associated virus control group（WT+AAV-GFP group）,APP/PS1 mice+GFP adeno-associated virus control group（APP/PS1+AAV-GFP group）and APP/PS1 mice+adeno-associated virus overexpressing circ-Bptf group（APP/PS1+AAV-circ-BPT group）F group),q RT-PCR was used to detect the overexpression efficiency of circ-Bptf in the hippocampus of APP/PS1 mice.Y maze,novel object recognition and Morris water maze tests were used to detect the changes in learning and memory ability of APP/PS1 mice after overexpression of AAV-circ-Bptf.1.3 Effect of AAV-circ-Bptf overexpression on the density and morphology of dendritic spines and the protein expression of Drebrin,PSD95 and p62 in the hippocampus of APP/PS1 mice.Golgi staining was used to observe the density and morphology of dendritic spines in the CA1 region of hippocampus.Immunohistochemical staining was used to observe the expression changes of synaptic plasticity related proteins Drebrin and PSD95 in the CA1 region of hippocampus.At the molecular level,the expressions of Drebrin,PSD95 and p62proteins were detected by Western Blot.q RT-PCR was used to detect the expression of mi R-138-5p and p62 m RNA.2.Bioinformatics analysisThrough bioinformatics analysis websites such as"RNAhybrid"and"Target Scan",the circ-Bptf/mi R-138-5p/p62 m RNA interaction network was predicted.3.Cellular level3.1 Fluorescence in situ hybridization was used to verify the subcellular localization of circ-Bptf in HT22 cells.3.2 In HEK 239A cells,the dual luciferase reporter gene experiment was used to verify the targeted binding of circ-Bptf/mi R-138-5p/p62.3.3 After knocking down circ-Bptf in HT22 cells,the expression changes of circ-Bptf,mi R-138-5p and p62 were detected.HT22 cells were divided into two groups:negative control group（si-NC）and circ-Bptf knockdown group（si-circ-Bptf）.The knockdown efficiency of circ-Bptf and the expression of mi R-138-5p were detected by q RT-PCR.The expression of p62 was detected by q RT-PCR and Western Blot.3.4 After overexpression of circ-Bptf in HT22 cells,the expression changes of circ-Bptf,mi R-138-5p and p62 were detected.HT22 cells were divided into two groups:the empty vector control group（vec-NC）and the circ-Bptf overexpression group（vec-circ-Bptf）.The overexpression efficiency of circ-Bptf and the expression of mi R-138-5p were detected by q RT-PCR.The expression of p62 was detected by q RT-PCR and Western Blot.3.5 After overexpression of mi R-138-5p in HT22 cells,the expression of p62 was detected.HT22 cells were divided into two groups:a negative control group（NC mimics）and a mi R-138-5p overexpression group（mi R-138-5p mimics）.The expression of p62 was detected by q RT-PCR and Western Blot.3.6 Knockdown of circ-Bptf and inhibition of mi R-138-5p in HT22cells were used to detect the expression changes of p62.HT22 cells were divided into three groups:Negative control group（si-NC+NC inhibitor）,circ-Bptf knockdown+negative control group（si-circ-Bptf+NC inhibitor）and circ-Bptf knockdown+mi R-138-5p inhibition group（si-circ-Bptf+mi R-138-5p）The expression of p62 was detected by q RT-PCR and Western Blot.Results:1.The expression of circ-Bptf in hippocampus of APP/PS1 mice was significantly decreased.The results of Y maze test showed that compared with the WT group,the proportion of exploration time and the proportion of exploration distance in the novel arm of APP/PS1 group mice were significantly reduced.The results of novel object recognition test showed that compared with the WT group,APP/PS1 group mice had a significant reduction in the discrimination index of novel objects after 2 h and 24 h intervals.The results of Morris water maze test showed that compared with the WT group,the latency of APP/PS1 group mice was significantly prolonged on the 5th day,and the residence time in the target quadrant and the number of platform crossings of APP/PS1 group mice were also significantly reduced.q RT-PCR results showed that there was no significant change in the expression of circ-Bptf in the hippocampus of APP/PS1 mice at 3 and 6months of age,but the expression of circ-Bptf in the hippocampus of APP/PS1 mice decreased at 9 months of age,and the decrease was most significant at 12 months of age.2.Overexpression of circ-Bptf in the hippocampus can improve learning and memory impairment in APP/PS1 mice.q RT-PCR results showed that the expression of circ-Bptf in the hippocampus of APP/PS1+AAV-circ-Bptf group was significantly higher than that of WT+AAV-GFP group and APP/PS1+AAV-GFP group after hippocampal injection of circ-Bptf virus.The results of Y maze experiment showed that compared with the WT+AAV-GFP group,the proportion of exploration time and exploration distance in the novel arm of the APP/PS1+AAV-GFP group mice was significantly reduced,and the proportion of exploration time and exploration distance in the novel arm of the APP/PS1+AAV-circ-Bptf group mice was significantly reduced.Compared with APP/PS1+AAV-GFP group,the mice in APP/PS1+AAV-circ-Bptf group had a significant increase in the proportion of exploration time and distance in the novel arm.The results of novel object recognition test showed that compared with the WT+AAV-GFP group,the APP/PS1+AAV-GFP group had a significant reduction in the recognition index of novel objects after 2 h and24 h interval,while the APP/PS1+AAV-circ-Bptf group had no significant change in the recognition index of novel objects after 2 h interval.After an interval of 24 hours,the discrimination index of new objects decreased significantly.Compared with APP/PS1+AAV-GFP group,APP/PS1+AAV-circ-Bptf group mice showed a significant increase in the discrimination index of novel objects.Morris water maze test showed that compared with WT+AAV-GFP group,APP/PS1+AAV-GFP group mice had significantly prolonged latency,and significantly reduced residence time in the target quadrant and the number of platform crossings on day 5.The latency of APP/PS1+AAV-circ-Bptf group was significantly prolonged on the 5th day,and the residence time in the target quadrant and the number of platform crossings were reduced.Compared with APP/PS1+AAV-GFP group,the latency of APP/PS1+AAV-circ-Bptf group was significantly shortened on the 5th day,and the residence time in the target quadrant and the number of platform crossings were significantly increased.3.Hippocampal overexpression of circ-Bptf increased the density of hippocampal dendritic spines and the proportion of mushroom dendritic spines in APP/PS1 mice,and promoted the expression of synaptic plasticity proteins Drebrin and PSD95.Golgi staining results showed that compared with WT+AAV-GFP group,the density of dendritic spines and the proportion of mushrot-shaped dendritic spines in the hippocampal CA1 region of APP/PS1+AAV-GFP group mice were significantly reduced,while the density of dendritic spines and the proportion of mushrot-shaped dendritic spines in the hippocampal CA1 region of APP/PS1+AAV-circ-Bptf group did not change significantly.Compared with APP/PS1+AAV-GFP group,the density of dendritic spines and the proportion of mushroom-shaped dendritic spines in the hippocampal CA1 region of APP/PS1+AAV-circ-Bptf group increased.Western Blot results showed that compared with the WT+AAV-GFP group,the expression of Drebrin and PSD95 protein in the hippocampus of APP/PS1+AAV-GFP group mice was significantly decreased.There was no significant change in Drebrin and PSD95 protein in hippocampus of APP/PS1+AAV-circ-Bptf group.Compared with APP/PS1+AAV-GFP group,the expressions of Drebrin and PSD95 protein in the hippocampus of APP/PS1+AAV-circ-Bptf group were increased in different degrees.The results of immunohistochemical staining showed that compared with the WT+AAV-GFP group,the expressions of Drebrin and PSD95proteins in the hippocampal CA1 region of APP/PS1+AAV-GFP group were significantly decreased.The expression of Drebrin protein in the hippocampal CA1 region of APP/PS1+AAV-circ-Bptf group did not change significantly,while the expression of PSD95 protein decreased.Compared with APP/PS1+AAV-GFP group,the expression of Drebrin and PSD95 protein in the hippocampal CA1 region of APP/PS1+AAV-circ-Bptf group was increased.4.circ-Bptf targets p62 expression by competitive binding to mi R-138-5p.4.1 Fluorescence in situ hybridization showed that circ-Bptf was mainly localized in the cytoplasm.4.2 circ-Bptf binds to mi R-138-5pIn HEK-293A cells,the results of dual luciferase reporter gene assay showed that the relative luciferase activity was significantly decreased after co-transfection of circ-Bptf wild type and mi R-138-5p mimic.In HT22 cells,after knocking down circ-Bptf,q RT-PCR results showed that the expression of mi R-138-5p in the si-circ-Bptf group was significantly increased compared with the si-NC group.In HT22 cells,after overexpression of circ-Bptf,q RT-PCR results showed that the expression of mi R-138-5p in the vec-circ-Bptf group was significantly reduced compared with the vec-NC group.4.3 mi R-138-5p targeted p62In HEK 293A cells,the results of dual luciferase reporter gene assay showed that the relative luciferase activity was significantly decreased after co-transfection of p62 wild type and mi R-138-5p mimic.After overexpression of mi R-138-5p in HT22 cells,q RT-PCR and Western Blot results showed that the expression of p62 m RNA and p62protein in the mi R-138-5p mimics group was decreased compared with that in the NC mimics group.4.4 circ-Bptf targets p62 expression by competitive binding to mi R-138-5pAfter knocking down circ-Bptf in HT22 cells,q RT-PCR results showed that the expression of circ-Bptf in the si-circ-Bptf group was decreased compared with that in the si-NC group.The results of q RT-PCR and Western Blot showed that the expression of p62 m RNA and protein in the si-circ-Bptf group was decreased compared with that in the si-NC group.After overexpression of circ-Bptf in HT22 cells,q RT-PCR results showed that the expression of circ-Bptf in the vec-circ-Bptf group was increased compared with that in the vec-NC group.The results of q RT-PCR and Western Blot showed that the expression of p62 m RNA and protein in the vec-circ-Bptf group was increased compared with that in the vec-NC group.When knocking down circ-Bptf and inhibiting mi R-138-5p in HT22cells,q RT-PCR and Western Blot results showed that compared with si-NC+NC inhibitor,The expression of p62 m RNA and p62 protein in the si-circ-Bptf+NC inhibitor group was significantly decreased,and the expression of p62m RNA in the si-circ-Bptf+mi R-138-5p inhibitor group was significantly increased,and the expression of p62 protein was decreased.Compared with the si-circ-Bptf+NC inhibitor group,the expression of p62 m RNA and p62 protein in the si-circ-Bptf+mi R-138-5p inhibitor group was increased.In APP/PS1 mice,after stereotactic injection of AAV-circ-Bptf into the hippocampus,q RT-PCR results showed that the expression of circ-Bptf was significantly decreased and the expression of mi R-138-5p was significantly increased in the hippocampus of APP/PS1+AAV-GFP mice compared with the WT+AAV-GFP group.The expression of p62 m RNA was significantly decreased,and the expression of circ-Bptf and p62m RNA in the hippocampus of APP/PS1+AAV-circ-Bptf group was significantly increased.Compared with APP/PS1+AAV-GFP group,the expression of circ-Bptf in the hippocampus of APP/PS1+AAV-circ-Bptf group was significantly increased,the expression of mi R-138-5p was significantly decreased,and the expression of p62 m RNA was significantly increased.Western Blot results showed that compared with the WT+AAV-GFP group,the expression of p62 protein in the hippocampus of APP/PS1+AAV-GFP group mice was significantly reduced,and the expression of p62 protein in the hippocampus of APP/PS1+AAV-circ-Bptf group mice was reduced.Compared with APP/PS1+AAV-GFP,the expression of p62 protein in the hippocampus of APP/PS1+AAV-circ-Bptf group was significantly increased.Conclusions:1.APP/PS1 mice aged 12 months showed learning and memory impairment,and the expression of circ-Bptf in the hippocampus was significantly reduced.2.Overexpression of circ-Bptf can improve learning and memory disorders and synaptic plasticity disorders in APP/PS1 mice.3.circ-Bptf can improve learning and memory disorders and synaptic plasticity in APP/PS1 mice by competitively binding to mi R-138-5p to up-regulate the expression of p62. |
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