1. Trafficking and processing of APP  -secretase

2. Intracellular trafficking of APP: relation to its physiological function?

3. APP NH 2 5A3/1G7 TMD APP localizes to the plasma membrane, Golgi and endosomes Pastorino, unpublished data

4. APP NH 2 TMD C-term APP Ab Staining for C-term domain of APP is detected also in the nucleus Pastorino, unpublished data

5. COOH APP NH 2 5A3/1G7 TMD APP internalizes from the plasma membrane into intracellular compartments, endosomes and Golgi Koo and Squazzo, 1994

6. Protein trafficking and endocytosis

7. APP co-localizes with the endosomes Pastorino et al., 2006

8. Because, the intracellular localization of APP INFLUENCES the production of  Amyloid peptide Why we want to study the trafficking of APP?

9. Processing of APP  -secretase Protective non-amyloidogenic pathway Pathogenic amyloidogenic pathway APP TMD s  APPs C83 s  APPs C99  -secretase NH2 COOH AA AICD p3 AICD  -secretase  -secretase  -secretase  -secretase  -secretase

10. Intracellular compartments and processing of APP  -secretase activity: in the plasma membrane (where metalloproteases, known to have like TACE and ADAM10/ADAM17, reside).  -secretase activity: mostly in the endosomes, possible also in the ER and Golgi  -secretase activity: mostly in the ER, also in lysosomes and possible at the plasma membrane (still under debate).

11. Trafficking of APP  -secretase activity  -secretase activity  -secretase activity  APP C83 APP C99  APPs APP AICD C99 AA

12. APP retained @ plasma membrane = Internalization of full length APP = GOOD!!  BAD!!! Protective non-amyloidogenic processing pathogenic amyloidogenic processing

13. Products dowstream of non-amyloidogenic processing:  APPs: soluble stub of APP deriving from the  -secretase cleavage : possible neurotrophic function p3: c-terminal truncated portion of the sequence of b-amyloid, deriving from the subsequent action of  - and  -secretase. DOES NOT aggregate. Unknown function. AICD: APP Intra Cellular Domain, deriving from the cleavage of  - secretase. Known regulation of transcriptional activity.

14. Products downstream of the amyloidogenic processing:  APPs: soluble stub of APP deriving from the  -secretase cleavage : unknown function C99: c-terminal stub containing the entire intact sequence of the  - amyloid peptide, deriving from the action of  -secretase. It is the substrate from where  -amyloid peptides derive.  -Amyloid peptides: generated by the subsequent action of  - and  - secretases. At very low concentration could be neurotrophic, however, when forming aggregates they are VERY TOXIC and lead to the formation of the core of the  -amyloid plaque in AD AICD: APP Intra Cellular Domain, deriving from the cleavage of  - secretase. Known regulation of transcriptional activity.

16. Loss of non-amyloidogenic activity as a possible way to develop AD?

17. Alzheimer’s pathology and depression

18. Selective Serotonin reuptake inhibitors (SSRI) reduce ISF Abeta…

19. …and activate protective pathways

20. Chronic SSRI treatment reduces the load of Abeta plaques in AD mice

21. Chronic SSRI treatment reduces the load of Abeta peptides in AD mice…

22. …and increases alpha-secretase activity

23. Use of antidepressant associates to reduced PIB uptake in humans

24. Activation of serotoninergic receptors leads to increased non-amyloidogenic pathway

25. Activation of non-amyloidogenic pathway as protective from AD?

26. Sirtuin: deacetylation and control on protein transcription

27. Sirtuins are involved in different diseases

28. Sirtuins may protect from AD

29. Sirtuins levels are reduced in aging Is sirtuins activity lost in AD? Could their activity be protective?

30. SIRT1 Tg AD mice show reduced plaque and Abeta load

31. Sirt1 expression regulates non-amyloidogenic processing of APP in AD mice

32. Sirt1 expression regulates levels of the  -secretase ADAM10 in AD mice both as protein….

33. …and as mRNA

34. SIRT1 regulates ADAM10 expression by deacetylating Retinoic Acid Receptor beta (RAR 

35. Levels of the transcription factor HES1 are regulated by SIRT1

36. Model: SIRT1 controls expression of ADAM10

37. The aspartyl protease BACE  -Amyloid cleaving enzyme

38. BACE is expressed mostly in the brain Vassar et al., 1999

39. In the cell, BACE localizes to Golgi apparatus and Endosomes

40. 1-In vitro, BACE is mostly active at an acidic pH range between 4.5- 5.5. 2-BACE is supposed to be mostly active in the endosomes, due to BACE co-localization and to the acidic pH of these organelles. Although in vivo, interaction between BACE and APP was observed at the plasma membrane and in the endosomes, in cell culture, BACE was active also in the ER and in the Golgi apparatus. BACE activity

42. It was previously reported that BACE interacts with GGAs in yeast two hybrid system GGA1, 2 and 3 are monomeric adaptors that are recruited to the trans-Golgi network. GGAs’ function relates to regulation of the trafficking and degradation of proteins GGA consists of 4 distinct domains: VHS domain that binds DxxxLL residue in proteins GAT domain which binds Arf:GTP Hinge region to recruit clathrin A gamma adapton ear homology domain Tesco et al., Neuron. 2007 Jun 7;54(5):721-37.

43. BACE co-localizes and is degraded within lysosomes Koh et al., 2005

44. Tesco et al., Neuron. 2007 Jun 7;54(5):721-37. APP contains caspase cleavage sites in its sequence However, although apoptosis increases C99 and A  levels, this effects do not depend on caspase-mediated cleavage of APP (Tesco et al., 2003).

45. Does apoptosis lead to increased amyloidogenic processing of APP up-regulating BACE?

46. In this paper: Apoptosis increases levels of BACE and C99 in different cell lines These effects are reverted by the apoptosis inhibitor zVAD. Apoptosis regulates levels of BACE affecting its degradation not its synthesis, suggesting a post-translational mechanism. During apoptosis GGA3 is cleaved generating a Dominant Negative fragment that further affects BACE trafficking and degradation.

47. Tesco et al., Neuron. 2007 Jun 7;54(5):721-37. Apoptosis increases levels of C99…..

48. Tesco et al., Neuron. 2007 Jun 7;54(5):721-37. …and BACE

49. Apoptosis regulates levels of BACE affecting its degradation not its synthesis The BACE trafficking molecule GGA3 is cleaved during apoptosis A dominant negative truncated form of GGA3 is generated under apoptotic stimuli, increasing levels of A  and BACE

50. What happens reducing the levels of GGA3?

51. Tesco et al., Neuron. 2007 Jun 7;54(5):721-37. GGA3 siRNA causes increase of BACE expression and accumulation of C99

52. Tesco et al., Neuron. 2007 Jun 7;54(5):721-37. Ischemic patients have increased levels of BACE in the brain…

53. Tesco et al., Neuron. 2007 Jun 7;54(5):721-37. And decreased levels of GGA3

54. Tesco et al., Neuron. 2007 Jun 7;54(5):721-37. AD patients have increased levels of BACE and decreased levels of GGA3 in the brain…

55. Apoptosis stabilizes BACE causing elevated  -secretase activity. This mechanism is regulated via the caspase-dependent cleavage of GGA3, that reduces the amount of BACE targeted to the lysosomes for degradation. AD, but not NAD patients show increased levels of BACE paralleled by decreased levels of GGA3.

56. Vassar, Neuron. 2007 Jun 7;54(5):671-3. Review. Model of BACE stabilization during apoptosis

57. BACE activity preceding AD? Brain Trauma?

58. Levels of BACE increase following Traumatic Brain Injury

59. BACE KO protects from Traumatic Brain injury

60. Hippocampal lesions are reduced in BACE KO

61. Can BACE trafficking contribute to amylodogenic pathology? Could BACE trafficking be targeted for therapeutic purposes?

62. Nature Reviews Molecular Cell Biology 10, 513-525 RAB5 involved in early endosomes formation

63. ARF6 regulates endocytosis

64. A dominant positive RAB5 mutant that leads to formation of large endosomes Question: is BACE localized to and active in RAB5 vesicles?

65. BACE colocalizes to and is active in RAB5 vesicles

66. BACE internalizes also independently on clathrin mediated endocytosis

67. Localization of BACE to RAB5 positive vesicles is independent on clathrin

68. BACE co-localizes with ARF6 cargo molecules

69. BACE colocalizes with ARF6-Q67L, a mutant that blocks the sorting of the vesicles to the early endosomes

70. Reduced levels of BACE at the early endosomes result in decreased Abeta: regulation of the ARF6 sorting pathway as a way to control BACE activity and reduce Abeta load

71. ARF6 regulates the sorting of intracellular BACE

72. Model: control of clathrin independent endocytosis to reduce BACE levels in the endosomes, thus to reduce BACE activity