Jürgen Kraus

1. Tolerance to Opioids and Cannabinoids: a Novel Role for MicroRNA Jürgen Kraus Summer School Bologna 27.6.2012

2. Tolerance Pharmacology: Decreased effectiveness of a drug upon repeated application >>> higher doses

3. Tolerance

4. Tolerance Pharmacology: Decreased effectiveness of a drug upon repeated application >>> higher doses Decreased amount of functional membrane-bound receptors for this drug

5. Tolerance Decreased amount of membrane-bound receptors 1. Phosphorylation/internalization of receptors resulting in decreased effectiveness 2. Downregulation of receptors resulting in deceased amounts

6. P P Ligand-Induced Phosphorylation and Internalization Localization in Cellmembrane Ligand-Induced Phosphorylation Internalization Recycling Degradation

7. P P Localization and Ligand-Induced Regulation of µ-Opioid Receptors in Jurkat T Cells

8. Tolerance - „internalizing drugs“ (e. g. fentanly or methadone) cause the same tolerance as „non-internalizing drugs“ (e. g. morphine) - tolerance develops within a range of minimum 12 hours up to several days

9. Tolerance Decreased amount of membrane-bound receptors 1. Phosphorylation/internalization of receptors resulting in decreased effectiveness 2. Downregulation of receptors resulting in deceased amounts

10. DNA > RNA > Protein

11. MOR mRNA is NOT Downregulated by Opioids

12. microRNA DNA > RNA > Protein

13. MOR mRNA is NOT Downregulated by Opioids

14. Tolerance to Opioids and Cannabinoids Involves MicroRNA

17. ? Does µRNA regulate MOR and CB1

18. µ Opioid Receptor Cannabinoid Receptor 1 Structure Effects Expression Cross-Tolerance

19. Functional Analysis of MOR and CB1 - Stimulation of cells with MOR and CB1 ligands results in phosphorylation of the mitogen activated protein kinase p42/44 - Stimulation of cells with MOR and CB1 ligands results in a decrease in intracellular cAMP due to inhibition of adenylyl cyclase

20. µRNA MOR CB1 µRNA miR-98, let-7a, let-7d, miR-23b MOR CB1

21. Overexpression of µRNA Inhibits Ligand-Induced Formation of Phospho-MAPK µRNA MOR CB1 SH SY5Y

22. µRNA MOR CB1 Overexpression of µRNA Inhibits Ligand-Induced Inhibition of cAMP Formation

23. µRNA MOR CB1 Overexpression of µRNA Inhibits Ligand-Induced Inhibition of cAMP Formation

24. Summary miR-98, let-7a, let-7d MOR

25. µRNA MOR CB1 Overexpression of µRNA Inhibits Ligand-Induced Inhibition of cAMP Formation

26. µRNA MOR CB1 Overexpression of µRNA Inhibits Ligand-Induced Inhibition of cAMP Formation

27. Summary miR-98, let-7a, let-7d, miR-23b MOR CB1

28. Summary let-7d MOR CB1

29. And Now for Something (Completely) Different ...

30. And Now for Something (Completely) Different ... - Tolerance - Crosstolerance (some colums are missing, but you will see them later)

31. And Now for Something (Completely) Different ... - Tolerance - Crosstolerance

32. Upregulation of µRNA in Response to Opioids and Cannabinoids Must be shown by µRNA Sequencing

33. ? How can we demonstrate functional induction of let7-d by opioids and cannabinoids

35. Upregulation of µRNA in Response to Opioids and Cannabinoids

36. Upregulation of µRNA in Response to Opioids and Cannabinoids

37. ? What about tolerance, when we inhibit let7-d µRNA

39. Human let-7d Inhibits Human and Rat MOR Expression

40. MAEA morphine let-7d MOR CB1 MAEA morphine let-7d MOR CB1

41. MAEA morphine let-7d MOR CB1 µRNA Might is Likely to Produce Tolerance to Opioids and Cannabinoids

42. µRNA Might is Likely to Produce Tolerance to Opioids and Cannabinoids

43. So far, so good, but tolerance is not a cellular event (even when many people say so)

44. Can Antagomirs Against let-7d Inhibit Tolerance to Opioids in Mice? Antagomirs = Antagonists to µRNAs RNA oligonucleotides protected chemically against rapid degradation Are linked to cholesterol to get into the cells Designed for use in animals (and humans!) Were applied icv in our experiments 4 times 8µg (in 4 µl) were injected, 3 days before testing analgesia

45. Antagomirs against let-7d Inhibit Tolerance to Opioids in Mice

46. Summary miR-98, let-7a, let-7d, miR-23b MOR CB1

47. Summary let-7d MOR CB1

48. Summary MAEA morphine let-7d MOR CB1

49. Summary MAEA morphine let-7d MOR CB1

50. Summary MAEA morphine let-7d MOR CB1