| Part Ⅰ Is there circadian dysfunction in the PD model induced by alpha-synuclein over-expression?Objective:In the clinical research and basic studies,there is increasing evidence that sleep disorders,abnormal 24-hour patterns of heart rate,and alterations of clock genes expression are commonly observed in Parkinson’s disease(PD).These hint us that circadian dysfunction is manifested in PD,which is characterized with the deposit of Lewy body and the loss of the dopaminergic neurons in the substantia nigra.Moreover,circadian disruption can also be found in other synucleinopathy,including the Lewy body’s dementia and multiple system atrophy.However,whether the aggregation of α-synuclein(SNCA)is related with the circadian disruption is unclear.Thus,this research was to investigate whether the circadian dysfunction can be induced by over-expression of α-synuclein.Methods:The 3-month old and 5-month old transgenic mice with SNCAA53-T over－expression and littermates are applied in our studies.And the free-running system was used to detect the period of rest-activity cycle.The activity amount per hour was measured by the energy metabolism cage system.In the molecular level,western blotting and real－time quantitative polymerase chain reaction(QPCR)were to determine the alterations of the mRNA and protein of clock genes,respectively.Results:Both in the 3-month-old and 5-month-old mice,the period of rest-activity cycle did not differ in the SNCAA53T mice from that of wild-type littermates.But the energy metabolism cage test showed that the 24-hour activity pattern in the SNCAA53T mice was different from that of wild-type littermates.In addition to the change of behavior test,the BMAL1 mRNA tended to decline in the 3-month-old SNCAA53T mice.And in the 5-month-old SNCAA53rmice,the BMAL1 mRNA was significantly lower than that in the age-matched litteramates.Likely,the western blotting also confirmed that the expression of BMAL1 protein decreased in the 5-month-old SNCAA53rmice.In vitro,the expression of BMAL1 protein and mRNA was inhibited induced by the SNCA expression in the PC 12 cell line.Conclusion:Our study indicated that the biorhythm disruption and abnormal expression pattern of clock genes could be induced by SNCA over-expression.Part Ⅱ The underlying mechanism of decreased BMAL1 induced by alpha-synuclein over-expressionObjective:The previous data indicated that the circadian dysftunction could be induced by the SNCA over-expression,along with the decreased level of BMAL1.In addition,the mRNA level of BMAL1 also is observed to be lower in PD patients than that in the age matched PD patients.Nevertheless,the underlying mechanism has not been uncovered.Therefore,we try to find how the SNCA aggregation is related with the abnormal BMAL1 expression in this part.Methods:The stable PC 12 cell with SNCA and vector over-expression was obtained by the lenti-virus infection method.After the cycloheximide and Dactinomyxin D treatment,the BMAL1 protein degradation rate was measured by western blotting and the stability of BMAL1 mRNA was determined by QPCR respectively.Next,bisulfite transformation method was applied to analyze the methylation of BMAL1 promotor and the DNA methyltransferase expression was detected by qPCR.At last,the miR-155 level was checked and its inhibitor was used to further confirm whether BMAL1 was regulated by the miR-155.Results:When compared with the Vector group,the degradation of BMAL1 did not change after the SNCA over-expression,but the mRNA of BMAL1 was lower in the SNCA over-expression group.In addition,the methylation level of the BMAL1 promoter remained and the DNMTs expression did not differ between the two group.Importantly,we found the stability of BMAL1 mRNA decreased,especially at 3 hour after the Dactinmycin treatment.Furthermore,we found that miR-155 increased induced by SNCA over-expression and the decreased BMAL1 level could be partially reversed by the inhibitor of miR-155.Conclusion:Our study indicated that the abnormal expression pattern of clock genes associated with SNCA over-expression may be due to the decreased unstability of BMAL1 mRNA resulted by the miR-155. |
