The Study On Behavioral Characteristics And Mechanism Of Circadian Rhythm Disturbance In Transgenic Mice Of Alzheimer’s Disease

Objective:To observe the behavioral characteristics of the circadian rhythm in male and female PS1_M146V/APP_Swe/tauP301 L triple-transgenic model mice（3xTg-AD mice）at different age by wheel running behavioral test,and investigate the changes of per1 and per2 gene expression in suprachiasmatic nucleus（SCN）tissues,so as to provide experimental evidence for the characteristics and mechanism of circadian rhythm disturbance in the AD pathogenesis.Methods:3-to 9-month-old C57BL/6 mice and 3xTg-AD mice were housed individually in cages equipped with running wheels.Each mouse was entrained to 12 h light/12 h dark cycle（LD）cycle for 7 days and then transferred to constant darkness（DD）for12 days.The free-running period,average activity per hour and circadian amplitude were examined by using voluntary wheel-running test,with data collected by Vital View system and analyzed by ActiView system.After the behavioral tests completed,mice were entrained to 12 h light/12 h dark（LD）cycle for another 7 days.Then,the mice were decapitated and the expression of per1 or per2 gene in SCN at ZT0,ZT4,ZT8,ZT12,ZT16 and ZT20 was detected by real-time PCR technique.Results:（1）The endogenous and exogenous circadian rhythms disturbance in 3xTg-AD mice was gradually aggravated with the increase of age.In 12 h light/12 h dark cycle（LD）environment,3-month-old 3xTg-AD mice showed obvious locomotor activity at night and almost inactivity during the day,indicating the existence of regular circadian rhythm.At 6-month-old,when the lights turned off,the locomotor activity of 3xTg-AD mice obviously increased,but activity pattern was dispersed.At the same time,the locomotor activity during the time lights turned on increased,indicating the circadian rhythm disorder.To 9-month-old 3xTg-AD mice,the locomotor activity significantly increased at day and activities scattered at night,indicating obvious exogenous circadian rhythm disorder.In constant darkness（DD）environment,the locomotor activity in 3-month-old 3xTg-AD mice was mainly focused on subjective night.The free-running period in 3-month-old male and female 3xTg-AD mice was（23.2 ± 0.1）h and（23.3 ± 0.1）h,respectively,significantly shorter than（23.5 ± 0.1）h and（23.5 ± 0.1）h in male and female C57BL/6 mice.At 6-month-old,the running wheel activity in 3xTg-AD mice was dispersed.The free running period of female3xTg-AD mice was（23.3 ± 0.2）h,but the boundary between activity and rest phase in male 3xTg-AD mice was difficult to distinguish,and the free running period could not be calculated.To 9-month-old 3xTg-AD mice,the activity pattern was disorganized,without clear boundary between activity and rest phase,and the free-running period was unavailable.（2）The circadian rhythm and the distribution of locomotor activity of 3xTg-AD mice in LD and DD environment decreased with the age increasing,and showing the gender difference.In the LD environment,the average activity of 3-month-old3xTg-AD mice at ZT12-ZT24 was significantly higher than that at ZT0-ZT12（P<0.001）,which was similar to C57BL/6 mice and existing regular circadian rhythm.However,although the average activity of 6-month-old 3xTg-AD mice at ZT12-ZT24 was also significantly higher than that at ZT0-ZT12（P<0.001）,the average activity in male and female 3xTg-AD mice at ZT12-ZT24 was significantly lower than that in C57BL/6 mice with the same gender（P<0.01）.In addition,compared to female mice,the average activity of male 3xTg-AD mice increased at ZT0-ZT12（P<0.01）anddecreased at ZT12-ZT24（P <0.05）.At 9-month-old,compared to C57BL/6 mice with the same gender,the average activity of male 3xTg-AD mice significantly increased at ZT0-ZT12（P<0.01）and decreased at ZT12-ZT24（P<0.001）,with the average activity of female 3xTg-AD mice only decreased at ZT12-ZT24（P<0.001）,but had no obvious alteration at ZT0-ZT12.In addition,there was no significant difference between female and male 3xTg-AD mice at bright and dark environment（P>0.05）.In the DD environment,the average activity in 3-month-old female 3xTg-AD mice during the subjective night was significantly higher than that during the subjective day（P<0.01）,but there was no significant difference of the average activity in male3xTg-AD mice during the subjective day and subjective night（P>0.05）.At the age of6-month-old,compared to female C57BL/6 mice,the average activity of female3xTg-AD mice at CT0-CT12 and CT12-CT24 all obviously decreased（P<0.001）.At the same time,the average activity of male 3xTg-AD mice at CT0-CT12 was similar to female 3xTg-AD mice and male C57BL/6 mice（P>0.05）,but the average activity of male 3xTg-AD mice at CT12-CT24 was significantly decreased（P<0.01）.To9-month-old male and female 3xTg-AD mice,the average activity was significantly lower than that in C57BL/6 mice with same gender during the subjective day and subjective night（P<0.05）,with the similar average activity between female and male3xTg-AD mice during the whole day.（3）The circadian amplitude of the 3xTg-AD mice in the LD and DD environment decreased with the age increasing,and the circadian amplitude showing gender difference.In LD environment,the circadian amplitude of 3-,6-and9-month-old male and female 3xTg-AD mice were（87.0 ± 13.4）counts/5 min,（25.9± 2.2）counts/5 min,（14.3 ± 2.0）counts/5 min and（72.7 ± 9.3）counts/5 min,（47.4 ±5.7）counts/5 min,（35.9 ± 6.8）counts/5 min,respectively.At the age of 6-and9-month-old,the amplitude in male and female 3xTg-AD mice significantly lower than that in C57BL/6 mice with the same gender,accompanying with the amplitude in male 3xTg-AD mice significantly lower than that in female mice.In constant darkness（DD）environment,the circadian amplitude in 3-,6-and 9-month-old male and female 3xTg-AD mice were（40.6 ± 4.1）counts/5 min,（37.0 ± 7.3）counts/5 min,（29.3 ± 3.9）counts/5 min and（56.3 ± 1.8）counts/5 min,（60.4 ± 9.6）counts/5 min,（41.3 ± 9.6）counts/5 min,respectively,which all significantly lower than that in age-matched C57BL/6 mice.In addition,there was no statistical difference between the male and female 3xTg-AD mice at different age.（4）Compared to C57BL/6 mice,the expression characteristic of per1 and per2 gene in SCN of 3xTg-AD mice was altered,and the expression level decreased with the age increasing.In the LD environment,the expression profile of per1 and per2 gene in the C57BL/6 mice was increased firstly and then decreased.The peak level of per1 and per2 mRNA expression in C57BL/6 was at ZT8,except the ZT12 showed in9-month-old male mice.However,although the profile of per1 gene expression in3-month-old 3xTg-AD mice was similar to C57BL/6 mice,the profile in 6-and9-month-old 3xTg-AD mice was decreased firstly and then increased.As to the per2 gene expression,the profile in 3-and 6-month-old 3xTg-AD mice was increased firstly and then decreased,but the tendency changed to decreased firstly and then increased in 9-month-old mice.In addition,the expression peak of per1 and per2 mRNA in 3xTg-AD mice all appeared at ZT12,showing a delay compared with C57BL/6 mice.On the other hand,the relative expression levels of per1 and per2 genes in 6-and 9-month-old male 3xTg-AD mice were lower than that in 3-month-old mice at ZT4,ZT8,ZT12 and ZT16（P<0.05）,and the expression level of per2 gene at ZT16 in 6-month-old female 3xTg-AD mice was lower than that in 3-month-old mice.In addition,the relative expression levels of per1 and per2 genes in 6-and9-month-old male 3xTg-AD mice were significantly lower than that in female mice with the same age at ZT0,ZT4,ZT8 and ZT20（P<0.05）.Conclusion:Both male and female 3xTg-AD mice showed mild endogenous circadian rhythm disorder at 3-month-old,and exogenous circadian rhythm disorder occurred at 6-month-old,with the stability of circadian rhythm significantly decreased from 6-month-old.At the same time,the disturbance of circadian rhythm and the decrease of circadian rhythm stability in3xTg-AD mice aggravated with age increasing,with the more serious impairment appeared in male mice.On the other hand,compared to C57BL/6 mice,the expression characteristics of per1 and per2 genes in male and female 3xTg-AD mice altered,showing the delay of expression peak and the decrease of expression level at specific time points.In addition,the expression level of per1 and per2 genes in 3xTg-AD mice decreased with the increase of age,and the decrease in male mice was more obvious.The results of the present study indicated that the behavioral circadian rhythm disorder observed in male and female 3xTg-AD mice might be related to the change of per1 and per2 gene expression.