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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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slide1
Tolerance to Opioids and Cannabinoids: a Novel Role for MicroRNA

Jürgen Kraus

Summer School Bologna 26.6.2014

slide2
Tolerance

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

slide4
Tolerance

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

Decreased amount of functional membrane-bound receptors for this drug

slide5
Tolerance

Decreased amount of membrane-bound receptors

1. Phosphorylation/internalization of receptors resulting in decreased effectiveness

2. Downregulation of receptors resulting in deceased amounts

slide6
P

P

Ligand-Induced Phosphorylation

and Internalization

Localization in Cellmembrane

Ligand-Induced Phosphorylation

Internalization

Recycling

Degradation

slide7
P

P

Localization and Ligand-Induced Regulation of

µ-Opioid Receptors in Jurkat T Cells

slide8
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

slide9
Tolerance

Decreased amount of membrane-bound receptors

1. Phosphorylation/internalization of receptors resulting in decreased effectiveness

2. Downregulation of receptors resulting in deceased amounts

slide12
microRNA

DNA > RNA > Protein

slide17
?

Does µRNA regulate MOR and CB1

slide18
µ Opioid Receptor

Cannabinoid Receptor 1

Structure

Effects

Expression

Cross-Tolerance

slide19
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

slide20
µRNA

MOR CB1

µRNA

miR-98, let-7a, let-7d, miR-23b

MOR CB1

slide22
µRNA

MOR CB1

Overexpression of µRNA Inhibits Ligand-Induced Inhibition of cAMP Formation

slide23
µRNA

MOR CB1

Overexpression of µRNA Inhibits Ligand-Induced Inhibition of cAMP Formation

slide24
Summary

miR-98, let-7a, let-7d

MOR

slide25
µRNA

MOR CB1

Overexpression of µRNA Inhibits Ligand-Induced Inhibition of cAMP Formation

slide26
µRNA

MOR CB1

Overexpression of µRNA Inhibits Ligand-Induced Inhibition of cAMP Formation

slide27
Summary

miR-98, let-7a, let-7d, miR-23b

MOR CB1

slide28
Summary

let-7d

MOR CB1

slide30
And Now for Something (Completely) Different ...

- Tolerance

- Crosstolerance

(some colums are missing, but you will see them later)

slide33
?

Does µRNA regulate MOR and CB1

?

Do opioids and cannabinoids regulate µRNA

slide36
Upregulation of Drosha and Argonaute2

in Response to Morphine

in Neuronal SH SY5Y Cells

slide37
Upregulation of Dicer and Drosha

in Response to MAEA

in U87 Glia Cells

slide38
Direct Upregulation of µRNA in Response to Opioids and Cannabinoids

Must be shown by µRNA Sequencing

slide39
?

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

slide42
?

What about tolerance, when we inhibit

let7-d µRNA

slide44
Human let-7d Inhibits

Human and Rat MOR Expression

slide45
MAEA

morphine

let-7d

MOR CB1

MAEA

morphine

let-7d

MOR CB1

slide46
MAEA

morphine

let-7d

MOR CB1

µRNA is Likely to Produce Tolerance to Opioids and Cannabinoids

slide48
So far, so good,

but

tolerance is not a cellular event

(even when many people say so)

slide49
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

slide51
Summary

miR-98, let-7a, let-7d, miR-23b

MOR CB1

slide52
Summary

let-7d

MOR CB1

slide53
Summary

MAEA

morphine

let-7d

MOR CB1

slide54
Summary

MAEA

morphine

let-7d

MOR CB1

slide55
Summary

MAEA

morphine

let-7d

MOR CB1

slide56
tolerance

opioid-cannabinoid-crosstolerance

Summary

MAEA

morphine

let-7d

MOR CB1

slide57
Volker Höllt

Christine Börner

Elisa Martella

Dana Mayer

Karina Schäfer

Evelyn Kahl

Axel Becker

Doreen Heidemann

Giulia Fornabaio

Michele Scotto di Mase

Grazie

Danke

Thank you

Gracias

Grazie

Merci

Tesekkür



Obrigado

Bedankt

slide58
Antisense RNA and Anti-MicroRNA in Human Pharmacology

Jürgen Kraus

Summer School Bologna 26.6.2014

slide61
Miravirsen

New drug against HCV

Mechanism: anti miR122

Eradication of the virus

Monodrug or in Combination with ribavirine;

no interferone-therapy necessary

very well tolerated, very few adversive side effects

Drugs against Hepatitisvirus

rna interferenz
Möglichkeiten

Hemmung viraler Genexpression

Hemmung zellulärer Genexpression

Induktion zellulärer Genexpression?

Hemmung von Tumorgenen

RNA Interferenz
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