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

Jürgen Kraus

Summer School Bologna 26.6.2014


Tolerance MicroRNA

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


Tolerance MicroRNA


Tolerance MicroRNA

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

Decreased amount of functional membrane-bound receptors for this drug


Tolerance MicroRNA

Decreased amount of membrane-bound receptors

1. Phosphorylation/internalization of receptors resulting in decreased effectiveness

2. Downregulation of receptors resulting in deceased amounts


P MicroRNA

P

Ligand-Induced Phosphorylation

and Internalization

Localization in Cellmembrane

Ligand-Induced Phosphorylation

Internalization

Recycling

Degradation


P MicroRNA

P

Localization and Ligand-Induced Regulation of

µ-Opioid Receptors in Jurkat T Cells


Tolerance MicroRNA

- „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


Tolerance MicroRNA

Decreased amount of membrane-bound receptors

1. Phosphorylation/internalization of receptors resulting in decreased effectiveness

2. Downregulation of receptors resulting in deceased amounts



MOR mRNA is NOT Downregulated MicroRNA

by Opioids


microRNA MicroRNA

DNA > RNA > Protein


MOR mRNA is NOT Downregulated MicroRNA

by Opioids



? MicroRNA

Does µRNA regulate MOR and CB1


µ Opioid Receptor MicroRNA

Cannabinoid Receptor 1

Structure

Effects

Expression

Cross-Tolerance


Functional Analysis of MOR and CB1 MicroRNA

- 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


µRNA MicroRNA

MOR CB1

µRNA

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

MOR CB1



µRNA Phospho-MAPK

MOR CB1

Overexpression of µRNA Inhibits Ligand-Induced Inhibition of cAMP Formation


µRNA Phospho-MAPK

MOR CB1

Overexpression of µRNA Inhibits Ligand-Induced Inhibition of cAMP Formation


Summary Phospho-MAPK

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

MOR


µRNA Phospho-MAPK

MOR CB1

Overexpression of µRNA Inhibits Ligand-Induced Inhibition of cAMP Formation


µRNA Phospho-MAPK

MOR CB1

Overexpression of µRNA Inhibits Ligand-Induced Inhibition of cAMP Formation


Summary Phospho-MAPK

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

MOR CB1


Summary Phospho-MAPK

let-7d

MOR CB1



And Now for Something (Completely) Different ... Phospho-MAPK

- Tolerance

- Crosstolerance

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


And Now for Something (Completely) Different ... Phospho-MAPK

- Tolerance

- Crosstolerance


Tolerance on a cellular level looks very much the same as Phospho-MAPK

the effect of µRNA overexpression


? Phospho-MAPK

Does µRNA regulate MOR and CB1

?

Do opioids and cannabinoids regulate µRNA



Upregulation of Drosha and Argonaute2 Morphine

in Response to Morphine

in Neuronal SH SY5Y Cells


Upregulation of Dicer and Drosha Morphine

in Response to MAEA

in U87 Glia Cells


Direct Upregulation of µRNA in Response to Opioids and Cannabinoids

Must be shown by µRNA Sequencing


? Cannabinoids

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



? Cannabinoids

What about tolerance, when we inhibit

let7-d µRNA


Human let-7d Inhibits Cannabinoids

Human and Rat MOR Expression


MAEA Cannabinoids

morphine

let-7d

MOR CB1

MAEA

morphine

let-7d

MOR CB1


MAEA Cannabinoids

morphine

let-7d

MOR CB1

µRNA is Likely to Produce Tolerance to Opioids and Cannabinoids



So far, so good, Cannabinoids

but

tolerance is not a cellular event

(even when many people say so)


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



Summary Mice

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

MOR CB1


Summary Mice

let-7d

MOR CB1


Summary Mice

MAEA

morphine

let-7d

MOR CB1


Summary Mice

MAEA

morphine

let-7d

MOR CB1


Summary Mice

MAEA

morphine

let-7d

MOR CB1


tolerance Mice

opioid-cannabinoid-crosstolerance

Summary

MAEA

morphine

let-7d

MOR CB1


Volker Höllt Mice

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


Antisense RNA and Anti-MicroRNA in Human Pharmacology Mice

Jürgen Kraus

Summer School Bologna 26.6.2014



Miravirsen Mice

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 Mice

Hemmung viraler Genexpression

Hemmung zellulärer Genexpression

Induktion zellulärer Genexpression?

Hemmung von Tumorgenen

RNA Interferenz


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