1. Aβ-induced degradation of BMAL1 and CBP leads to circadian rhythm disruption in Alzheimer’s disease Molecular Neurodegeneration (19 March 2015 ； IF= 6.563 ； PMID ： 25888034) 王宁 2015.10.8

2. IMI CONFIDENTIAL www.xianimi.com Background 2 Aβ, Amyloid-beta SCN, Suprachiasmatic nucleus CBP, Creb-binding protein Per2, Period circadian protein homolog 2 BMAL1, Aryl hydrocarbon receptor nuclear translocator- like 生理节律相关的蛋白，时钟基因：

3. IMI CONFIDENTIAL www.xianimi.com Materials and Methods 3  Animal : 5XFAD mice (APP/PSEN1) AD 小鼠 APP K670N/M671L+I716V+V717I PSEN1 M146L+L286V  Cell: HT22 mouse hippocampal cells, Cos7 monkey kidney cells  Methods: Behavioral assay, Immunocytochemistry, Western blot, real-time PCR, Immunoprecipitation, Luciferase reporter assays, BiFC assay, ELISA.

4. IMI CONFIDENTIAL www.xianimi.com Results 4 1. Abnormal circadian behavior in 5XFAD mice

5. IMI CONFIDENTIAL www.xianimi.com Results 5 1. Abnormal circadian behavior in 5XFAD mice

6. IMI CONFIDENTIAL www.xianimi.com Results 6 2. The expression of molecular circadian clock genes, Bmal1, Cbp and Per2, are impaired in 5XFAD mice 与对照小鼠相比， AD 小鼠 SCN 中 Bmal1, Per2, Cbp mRNA 水平明显发生了改变。

7. IMI CONFIDENTIAL www.xianimi.com Results 7 2. The expression of molecular circadian clock genes, Bmal1, Cbp and Per2, are impaired in 5XFAD mice

8. IMI CONFIDENTIAL www.xianimi.com Results 8 2. The expression of molecular circadian clock genes, Bmal1, Cbp and Per2, are impaired in 5XFAD mice 与对照小鼠相比， AD 小鼠 SCN 中 Bmal1, Per2, Cbp 蛋白水平并不呈现振荡表 达。

9. IMI CONFIDENTIAL www.xianimi.com Results 9 3. Aβ induces the degradation of BMAL1 and CBP Aβ 作用之后，细胞内 BMAL1 和 CBP 蛋白水平的振荡表达被 减弱。

10. IMI CONFIDENTIAL www.xianimi.com Results 10 3. Aβ induces the degradation of BMAL1 and CBP

11. IMI CONFIDENTIAL www.xianimi.com Results 11 3. Aβ induces the degradation of BMAL1 and CBP Aβ 作用 24 h 之后，细胞内 BMAL1 和 CBP 蛋白水平的振荡表 达被减弱。

12. IMI CONFIDENTIAL www.xianimi.com Results 12 3. Aβ induces the degradation of BMAL1 and CBP Aβ 作用 24 h 之后，细胞内 BMAL1 和 CBP 蛋白的相 互作用被减弱。

13. IMI CONFIDENTIAL www.xianimi.com Results 13 4. The degradation of BMAL1 by Aβ involves the sumoylation of BMAL1 随着 Aβ 作用时间的增长， 细胞内 BMAL1 蛋白的水平 逐渐降低。

14. IMI CONFIDENTIAL www.xianimi.com Results 14 4. The degradation of BMAL1 by Aβ involves the sumoylation of BMAL1 Aβ 作用导致细胞内 BMAL1 蛋白的泛素化降解。

15. IMI CONFIDENTIAL www.xianimi.com Results 15 4. The degradation of BMAL1 by Aβ involves the sumoylation of BMAL1 沉默类泛素蛋白之后，减轻了 Aβ 诱导的 BMAL1 降解。

16. IMI CONFIDENTIAL www.xianimi.com Results 16 4. The degradation of BMAL1 by Aβ involves the sumoylation of BMAL1 BMAL1 突变体显著抑制了 Aβ 导致的 BMAL1 降解。

17. IMI CONFIDENTIAL www.xianimi.com Results 17 5. Aβ induces N-Cadherin CTF2-mediated degradation of CBP through the cleavage of N-Cadherin by γ-secretase N-Cad 的过表达抑制了 CBP 的水平。 Aβ 提高了 N- Cad CTF 的水平。

18. IMI CONFIDENTIAL www.xianimi.com Results 18 5. Aβ induces N-Cadherin CTF2-mediated degradation of CBP through the cleavage of N-Cadherin by γ-secretase γ 分泌酶抑制剂 Aβ 对 CBP 的降解作用是通 过 γ 分泌酶对 N-Cad 的裂解 来实现的。

19. IMI CONFIDENTIAL www.xianimi.com Results 19 6. Aβ modulates the expression of Per2 mRNA through Aβ-mediated degradation of BMAL1 and CBP Aβ 作用之后，细胞内 Per2 mRNA 水平显著变化。

20. IMI CONFIDENTIAL www.xianimi.com Results 20 6. Aβ modulates the expression of Per2 mRNA through Aβ-mediated degradation of BMAL1 and CBP

21. IMI CONFIDENTIAL www.xianimi.com Results 21 6. Aβ modulates the expression of Per2 mRNA through Aβ-mediated degradation of BMAL1 and CBP Aβ 对 Per2 的降解是通过对 BMAL1 和 CBP 的降解实现 的。

22. IMI CONFIDENTIAL www.xianimi.com Results 22

23. IMI CONFIDENTIAL www.xianimi.com Results 23

24. IMI CONFIDENTIAL www.xianimi.com Results 24

25. IMI CONFIDENTIAL Conclusions 1. Aβ may affect circadian rhythm in 5XFAD mice 2. 5XFAD mice have impairments in the regulation of circadian clock genes 3. Aβ impacts on the metabolic stabilities of BMAL1 and CBP proteins. 4. Aβ-induced BMAL1 degradation is due to the sumoylation of BMAL1 5. The cleavage of N-Cadherin by γ-secretase is the molecular mechanism underlying Aβ-induced degradation of CBP. 6. Aβ modulates the expression of Per2 mRNA through Aβ- mediated degradation of BMAL1 and CBP.

26. IMI CONFIDENTIAL Conclusions

27. Thank you for your attention !