DA-JC1 Improves Expression Of Proteins Associated With Learning And Memory By Antagonizing Aβ31-35-induced Circadian Rhythm Disorder

Circadian rhythm disorder occurs in early stage of Alzheimer’s disease（AD）and can aggravate the later decline of learning and memory.The abnormal deposition of amyloid-beta protein（Aβ）is a central role in the development of AD.Over-deposition of Aβinfluences the rhythmic expression of core clock genes and leads to decreased learning and memory ability.But there is no effective treatment for AD at present.Previous studies have shown that Type 2 diabetes mellitus（T2DM）has many similar pathophysiological features with AD,and common therapeutic drugs of T2DM,Glucagon-like peptide-1（GLP-1）and Glucose-dependent insulinotropic polypeptide（GIP）can cross the blood-brain barrier and improve AD by activating corresponding GLP-1R and GIPR which are expressed in brain.It has been known that the novel dual incretin receptor（GLP-1R/GIPR）agonist DA-JC1 has a neuroprotective effect.But whether it could improve the circadian rhythm disorder induced by Aβ31-35,and further improve the decline of proteins associated with learning and memory is unclear.In this study,we investigated that whether DA-JC1 could improve the circadian rhythm disorder and then improve the decline learning and memory ability induced by Aβ31-35 in C57BL/6 mice.We performed animal wheel-running experiments and found that free-running rhythm of mice was disrupted by intrahippocampal injection of Aβ31-35,and intraperitoneal injection of DA-JC1 significantly improved the disorder.Then we found that DA-JC1 pre-treatment ameliorated Aβ31-35-induced abnormal expression of Per1 in HT22 cells.On the other hand,DA-JC1 antagonised the decline of SYP and GAP-43 which are associated with learning and memory induced by Aβ31-35.Further we used lentiviral vector-transduced siRNAs to knock down per2 in HT22 cells and found that the rhythmic expression of per1 was abolished and the expression levels of SYP and GAP-43 proteins decreased.Thus we thought that DA-JC1 could improve the circadian rhythm disorder induced by Aβ31-35,and further ameliorate the decline of proteins associated with learning and memory.Part Ⅰ DA-JC1 improves circadian rhythm disorder in C57BL/6 mice and abnormal expression of clock genes in HT22 cells induced by Aβ31-35Objective:Investigate the effect of novel dual incretin receptor（GLP-1R/GIPR）agonist DA-JC1to circadian rhythm disorder induced by Aβ31-35.Methods:（1）The 6-8 week-old male C57BL/6 mice were divided into control group,Aβ31-35group,pre-DA-JC1 group and DA-JC1 group for wheel-running behavior experiment.（2）HT22 mouse hippocampal cells were adopted as the research objects.Cells were divided into control group,Aβ31-35 group,pre-DA-JC1 group and DA-JC1 group.We used MTT cytotoxicity assay to detect cell viability.RT-PCR was used to detect the expression of per1.Western blot was applied to examine the expression of PER1 protein at CT12.Results:（1）Compared with control group（23.54±0.07 h）,Aβ31-35 disturbed the circadian rhythm which exhibited significantly longer free running periods（23.80±0.06）and lower locotomor activity（635.68±47.63 counts/d）,whereas the disruption was significantly relieved with pre-treatment of DA-JC1（23.61±0.06 h,777.5±23.15 counts/d）.（2）Compared with 0μM Aβ31-35 group（100.0±3.6%）,5μM Aβ31-35 decreased the cell viability significantly（78.7±3.4%）.DA-JC1 can reduce the toxicity of Aβ31-35 in HT22cells（89.2±2.3%）.（3）Abnormal rhythmic expressions of Per1 were induced by Aβ31-35in HT22 cells which significantly decreased at CT12（per1 gene:0.58±0.04;PER1protein:0.74±0.07）compared with control group（per1 gene:1.00±0.09;PER1 protein:1.01±0.07）.While DA-JC1 pre-treatment can reverse the abnormal expression（per1 gene:0.79±0.11;PER1 protein:0.99±0.05）.Conclusion:DA-JC1 improved the circadian rhythm disorder in C57BL/6 mice and abnormal expression of Per1 in HT22 cells induced by Aβ31-35.PartⅡDA-JC1 antagonised the decline of SYP and GAP-43 which are associated with learning and memory induced by Aβ31-35Objective:Investigate the effect of DA-JC1 to the decline of SYP and GAP-43 which are associated with learning and memory induced by Aβ31-35 in HT22 cells.Methods:HT22 cells were divided into control group,Aβ31-35 group,pre-DA-JC1 group and DA-JC1 group randomly.Western blot was applied to examine the expression of SYP and GAP-43 proteins.Results:（1）Compared with control group,Aβ31-35 decreased expression of SYP（0.85±0.03vs 1.00±0.04）protein.While DA-JC1 pre-treatment can reverse the abnormal expression（1.01±0.03）.（2）The expression of GAP-43 was down-regulation by Aβ31-35（0.86±0.03 vs 1.00±0.03）and up-regulation by pretreatment of DA-JC1（0.97±0.06）.Conclusion:DA-JC1 improved the decline of SYP and GAP-43 protein in HT22 cells induced by Aβ31-35.PartⅢper2 scilencing induced circadian rhythm disorder led to the decline of SYP and GAP-43 proteinsObjective:Investigate the expression of SYP and GAP-43 proteins in HT22 cells which infected with shRNA to knock down per2 gene.Methods:（1）We infected HT22 cells with shRNA at a gradient multiplicity of infection（MOI）,and selected the optimal MOI.（2）We infected HT22 cells with shRNA to knock down per2in HT22 cells.Western blot was used to detect PER2 protein.（3）RT-PCR was applied to examine the expression of per1 at different circadian time points.（4）Western blot was applied to examine the expression of SYP and GAP-43 proteins in per2^-/-HT22 cells.Results:（1）Fluorescence value of cells was increased markedly at MOI of 60 compared with the least MOI group（P<0.05）.（2）The expression of PER2 protein was decreased significantly by shRNA（0.42±0.03 vs 1.00±0.09）.（3）The amplitude of per1 was much lower than control group（0.43±0.11 vs 1.00±0.18）.（4）Compared with control group,expression of SYP（0.80±0.02 vs 1.00±0.04）and GAP-43（0.81±0.03 vs 1.00±0.03）were reduced.Conclusion:The expression of SYP and GAP-43 proteins were declined in HT22 cells in which per2 gene was knock down.