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Jürgen Kraus

Tolerance to Opioids and Cannabinoids: a Novel Role for MicroRNA. Jürgen Kraus. Summer School Bologna 26.6.2014. Tolerance. Pharmacology: Decreased effectiveness of a drug upon repeated application >>> higher doses. Tolerance. Tolerance.

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Jürgen Kraus

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  1. Tolerance to Opioids and Cannabinoids: a Novel Role for MicroRNA Jürgen Kraus Summer School Bologna 26.6.2014

  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

  15. ? Does µRNA regulate MOR and CB1

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

  17. 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

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

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

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

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

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

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

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

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

  26. Summary let-7d MOR CB1

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

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

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

  30. Tolerance on a cellular level looks very much the same as the effect of µRNA overexpression

  31. ? Does µRNA regulate MOR and CB1 ? Do opioids and cannabinoids regulate µRNA

  32. Upregulation of Dicer, Drosha and Argonaute2 in Response to Morphine in Jurkat T Cells

  33. Upregulation of Drosha and Argonaute2 in Response to Morphine in Neuronal SH SY5Y Cells

  34. Upregulation of Dicer and Drosha in Response to MAEA in U87 Glia Cells

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

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

  37. Upregulation of µRNA in Response to Opioids and Cannabinoids

  38. ? 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 is Likely to Produce Tolerance to Opioids and Cannabinoids

  42. µRNA 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

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