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. 2010 Jul 8;466(7303):197-202.
doi: 10.1038/nature09202.

Striatal microRNA controls cocaine intake through CREB signalling

Jonathan A Hollander  1 , Heh-In Im, Antonio L Amelio, Jannet Kocerha, Purva Bali, Qun Lu, David Willoughby, Claes Wahlestedt, Michael D Conkright, Paul J Kenny
Affiliations

Striatal microRNA controls cocaine intake through CREB signalling

Jonathan A Hollander et al. Nature. .

Abstract

Cocaine addiction is characterized by a gradual loss of control over drug use, but the molecular mechanisms regulating vulnerability to this process remain unclear. Here we report that microRNA-212 (miR-212) is upregulated in the dorsal striatum of rats with a history of extended access to cocaine. Striatal miR-212 decreases responsiveness to the motivational properties of cocaine by markedly amplifying the stimulatory effects of the drug on cAMP response element binding protein (CREB) signalling. This action occurs through miR-212-enhanced Raf1 activity, resulting in adenylyl cyclase sensitization and increased expression of the essential CREB co-activator TORC (transducer of regulated CREB; also known as CRTC). Our findings indicate that striatal miR-212 signalling has a key role in determining vulnerability to cocaine addiction, reveal new molecular regulators that control the complex actions of cocaine in brain reward circuitries and provide an entirely new direction for the development of anti-addiction therapeutics based on the modulation of noncoding RNAs.

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Figures

Figure 1
Figure 1. Increased striatal miR-212 expression in extended access rats
a, Expression analysis revealed that striatal miR-212 levels were increased in rats with extended access to cocaine self-administration. b, Taqman assay verified that striatal miR-212 levels were increased in extended access rats (F3,23=6.6, P<0.01; *P<0.05, **P<0.01, statistically significant compared with the extended access group). c, Relative amounts of p-CREB in the dorsal striatum were quantified by densitometry (F2,6=6.8, P<0.05; *P<0.05 compared with control). The lower panel is a representative immunoblot of the increased striatal p-CREB expression. Data are presented as mean ± SEM.
Figure 2
Figure 2. Dissociable effects of striatal miR-212 oncocaine intake
a, Red circles in left panel are locations at which viral infusions were targeted in dorsal striatum. The right panel is representative immunochemistry staining from a Lenti-miR-212 rat. Green is GFP from virus; red is the astrocyte marker glial fibrillary acidic protein (GFAP); Ctx, cortex; CC corpus callosum; LV, lateral ventricle; DSt, dorsal striatum; yellow arrows highlight the injector track used to deliver virus. Insert is an x80 confocal image of a virus-infected neuron. b, FISH was used to visualize striatal miR-212 expression (shown in red) in Lenti-Control and Lenti-miR-212 rats. c, Striatal miR-212 overexpression reverses the long-term trajectory of cocaine-taking behavior in rats with extended access (Virus x Session: F13,130=3.0, P<0.001; *P<0.05, **P<0.01 compared with intake on the same day in Lenti-Control rats). d, LNA-antimiR-212 delivered into dorsal striatum increases cocaine intake in extended access rats (LNA x Session: F4,36=5.3, P<0.005; *P<0.05, **P<0.01, compared with intake on the same day in the LNA-Scrambled rats). Data are presented as mean ± SEM.
Figure 3
Figure 3. miR-212 amplifies CREB signaling
a, miR-212 potentiated CREB signaling engaged by forskolin (FSK),measured using an EVX-1 luciferase reporter (miRNA x FSK: F1,8=99.2, P<0.001). b, miR-212 potentiated forskolin-induced increases in Fos mRNA in HEK cells(miRNA x FSK: F1,8>1000, p<0.001). c, The effects of miR-212 on CREB signaling were abolished in GAL4-luciferase reporters containing phosphorylation-defective (ΔSer133) or TORC recruitment-defective (ΔR314) CREB mutants. d, A-CREB polypeptide abolished the effects of miR-212 on Fos mRNA expression. e, RNAi-mediated knockdown of TORC2 also abolished the effects of miR-212 on Fos mRNA expression. f, Protein levels of Fos were enhanced in dorsal striatum from Lenti-miR-212 rats with extended cocaine access compared with other groups. g, Striatal levels of Nurr1, another CREB-responsive gene, were also enhanced inLenti-miR-212 rats with extended access. Data are presented as mean ± SEM.
Figure 4
Figure 4. miR-212 stimulates core CREB signaling components
a, miR-212 potentiated cAMP accumulation in HEK cells (miRNA x FSK: F2,8=4.8, P<0.05). b, miR-212 also increased p-CREB and the related p-ATF-1, without altering total-CREB. c, Representative immunoblots of the increased TORC2 levels in HEK cells. d, Relative amounts of TORC2 in HEK cells expressing miR-212, miR-132 or vector (pMIF) were quantified by densitometry (*P<0.05 compared with pMIF). e, Representative immunoblots showing that miR-212 increased striatal levels of TORC1 and TORC2. f, The relative amounts of striatal TORC1 and TORC2 were quantified by densitometry (*P<0.05, **P<0.01, compared with Lenti-Control).Data are presented as mean ± SEM.
Figure 5
Figure 5. miR-212 amplifies CREB signaling through Raf-1
a, miR-212 activates Raf-1 signaling, reflected in increased levels of phosphorylated endogenous and exogenous (Raf-1-GFP) Raf-1 protein. b, Dominant-negative Raf-1 (DN-Raf-1) abolished the stimulatory effects of miR-212 on cAMP accumulation. c, DN-Raf-1abolished miR-212-induced increases in p-CREB. d, DN-Raf-1 also abolished miR-212-induced increases in TORC2. e, Enhancing Raf-1 signaling by pulsing cells with the Raf-1 inhibitor GW5074 potentiated cAMP accumulation. f, Potentiation of Raf-1 signaling increased p-CREB levels. g, Potentiation of Raf-1 signaling increased TORC2 levels. h, Knockdown of the Raf-1 repressor SPRED1, a target for miR-212, increased p-CREB levels. i, SPRED1 knockdown also increased TORC2 levels. Data are presented as mean ± SEM.
Figure 6
Figure 6. Striatal CREB:TORC signaling controls cocaine intake
a, Representative image of Lenti-TORC1-infected neurons (green) in the dorsal striatum. b, Representative high-magnification images of Lenti-TORC1 infected cells (green), demonstrating the viral-driven upregulation of TORC1 expression (red). Cell nuclei are shown in blue using DAPI staining. c, Relative amounts of TORC1in dorsal striatum were quantified by densitometry (*P<0.05). The lower panel is a representative immunoblot. d, Cocaine intake under extended access conditions is far lower in Lenti-TORC1 rats compared with Lenti-Controls(Virus x Session: F9,.90=2.9, P<0.001; *P<0.05, **P<0.001 compared with intake on the same day in Lenti-Control rats).Data are presented as mean ± SEM.
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