mTOR complex 1: a key player in neuroadaptations induced by drugs of abuse

The mammalian (or mechanistic) target of rapamycin (mTOR) complex 1 (mTORC1) is a serine and threonine kinase that regulates cell growth, survival, and proliferation. mTORC1 is a master controller of the translation of a subset of mRNAs. In the central nervous system mTORC1 plays a crucial role in mechanisms underlying learning and memory by controlling synaptic protein synthesis. Here, we review recent evidence suggesting that the mTORC1 signaling pathway promotes neuroadaptations following exposure to a diverse group of drugs of abuse including stimulants, cannabinoids, opiates, and alcohol. We further describe potential molecular mechanisms by which drug-induced mTORC1 activation may alter brain functions. Finally, we propose that mTORC1 is a focal point shared by drugs of abuse to mediate drug-related behaviors such as reward seeking and excessive drug intake, and offer future directions to decipher the contribution of the kinase to mechanisms underlying addiction. Recent studies suggesting that exposure to diverse classes of drugs of abuse as well as exposure to drug-associated memories lead to mTORC1 kinase activation in the limbic system. In turn, mTORC1 controls the onset and the maintenance of pathological neuroadaptions that underlie several features of drug addiction such as drug seeking and relapse. Therefore, we propose that targeting mTORC1 and its effectors is a promising strategy to treat drug disorders

GPCR and Alcohol-Related Behaviors in Genetically Modified Mice

International audienceG protein-coupled receptors (GPCRs) constitute the largest class of cell surface signaling receptors and regulate major neurobiological processes. Accordingly, GPCRs represent primary targets for the treatment of brain disorders. Several human genetic polymorphisms affecting GPCRs have been associated to different components of alcohol use disorder (AUD). Moreover, GPCRs have been reported to contribute to several features of alcohol-related behaviors in animal models. Besides traditional pharmacological tools, genetic-based approaches mostly aimed at deleting GPCR genes provided substantial information on how key GPCRs drive alcohol-related behaviors. In this review, we summarize the alcohol phenotypes that ensue from genetic manipulation, in particular gene deletion, of key GPCRs in rodents. We focused on GPCRs that belong to fundamental neuronal systems that have been shown as potential targets for the development of AUD treatment. Data are reviewed with particular emphasis on alcohol reward, seeking, and consumption which are behaviors that capture essential aspects of AUD. Literature survey indicates that in most cases, there is still a gap in defining the intracellular transducers and the functional crosstalk of GPCRs as well as the neuronal populations in which their signaling regulates alcohol actions. Further, the implication of only a few orphan GPCRs has been so far investigated in animal models. Combining advanced pharmacological technologies with more specific genetically modified animals and behavioral preclinical models is likely necessary to deepen our understanding in how GPCR signaling contributes to AUD and for drug discovery

Activation of the cAMP pathway induces RACK1-Dependent binding of β-Actin to BDNF promoter

RACK1 is a scaffolding protein that contributes to the specificity and propagation of several signaling cascades including the cAMP pathway. As such, RACK1 participates in numerous cellular functions ranging from cell migration and morphology to gene transcription. To obtain further insights on the mechanisms whereby RACK1 regulates cAMP-dependent processes, we set out to identify new binding partners of RACK1 during activation of the cAMP signaling using a proteomics strategy. We identified β-actin as a direct RACK1 binding partner and found that the association between β-actin and RACK1 is increased in response to the activation of the cAMP pathway. Furthermore, we show that cAMP-dependent increase in BDNF expression requires filamentous actin. We further report that β-actin associates with the BDNF promoter IV upon the activation of the cAMP pathway and present data to suggest that the association of β-actin with BDNF promoter IV is RACK1-dependent. Taken together, our data suggest that β-actin is a new RACK1 binding partner and that the RACK1 and β-actin association participate in the cAMP-dependent regulation of BDNF transcription

Activation of the cAMP Pathway Induces RACK1-Dependent Binding of β-Actin to BDNF Promoter.

RACK1 is a scaffolding protein that contributes to the specificity and propagation of several signaling cascades including the cAMP pathway. As such, RACK1 participates in numerous cellular functions ranging from cell migration and morphology to gene transcription. To obtain further insights on the mechanisms whereby RACK1 regulates cAMP-dependent processes, we set out to identify new binding partners of RACK1 during activation of the cAMP signaling using a proteomics strategy. We identified β-actin as a direct RACK1 binding partner and found that the association between β-actin and RACK1 is increased in response to the activation of the cAMP pathway. Furthermore, we show that cAMP-dependent increase in BDNF expression requires filamentous actin. We further report that β-actin associates with the BDNF promoter IV upon the activation of the cAMP pathway and present data to suggest that the association of β-actin with BDNF promoter IV is RACK1-dependent. Taken together, our data suggest that β-actin is a new RACK1 binding partner and that the RACK1 and β-actin association participate in the cAMP-dependent regulation of BDNF transcription

Direct interaction between scaffolding proteins RACK1 and 14-3-3 zeta regulates brain-derived Neurotrophic Factor ( BDNF) Transcription

RACK1 is a scaffolding protein that spatially and temporally regulates numerous signaling cascades. We previously found that activation of the cAMP signaling pathway induces the translocation of RACK1 to the nucleus. We further showed that nuclear RACK1 is required to promote the transcription of the brain-derived neurotrophic factor (BDNF). Here, we set out to elucidate the mechanism underlying cAMP-dependent RACK1 nuclear translocation and BDNF transcription. We identified the scaffolding protein 14-3-3ζ as a direct binding partner of RACK1. Moreover, we found that 14-3-3ζ was necessary for the cAMP-dependent translocation of RACK1 to the nucleus. We further observed that the disruption of RACK1/14-3-3ζ interaction with a peptide derived from the RACK1/14-3-3ζ binding site or shRNA-mediated 14-3-3ζ knockdown inhibited cAMP induction of BDNF transcription. Together, these data reveal that the function of nuclear RACK1 is mediated through its interaction with 14-3-3ζ. As RACK1 and 14-3-3ζ are two multifunctional scaffolding proteins that coordinate a wide variety of signaling events, their interaction is likely to regulate other essential cellular functions

The small G protein H-Ras in the mesolimbic system is a molecular gateway to alcohol-seeking and excessive drinking behaviors.: H-Ras in the NAc and alcohol consumption

International audienceUncontrolled consumption of alcohol is a hallmark of alcohol abuse disorders; however, the central molecular mechanisms underlying excessive alcohol consumption are still unclear. Here, we report that the GTP binding protein, H-Ras in the nucleus accumbens (NAc) plays a key role in neuroadaptations that underlie excessive alcohol-drinking behaviors. Specifically, acute (15 min) systemic administration of alcohol (2.5 g/kg) leads to the activation of H-Ras in the NAc of mice, which is observed even 24 h later. Similarly, rat operant self-administration of alcohol (20%) also results in the activation of H-Ras in the NAc. Using the same procedures, we provide evidence suggesting that the exchange factor GRF1 is upstream of H-Ras activation by alcohol. Importantly, we show that infection of mice NAc with lentivirus expressing a short hairpin RNA that targets the H-Ras gene produces a significant reduction of voluntary consumption of 20% alcohol. In contrast, knockdown of H-Ras in the NAc of mice did not alter water, quinine, and saccharin intake. Furthermore, using two-bottle choice and operant self-administration procedures, we show that inhibiting H-Ras activity by intra-NAc infusion of the farnesyltransferase inhibitor, FTI-276, produced a robust decrease of rats' alcohol drinking; however, sucrose consumption was unaltered. Finally, intra-NAc infusion of FTI-276 also resulted in an attenuation of seeking for alcohol. Together, these results position H-Ras as a central molecular mediator of alcohol's actions within the mesolimbic system and put forward the potential value of the enzyme as a novel target to treat alcohol use disorders

Disruption of alcohol-related memories by mTORC1 inhibition prevents relapse

a r t I C l e S Alcohol abuse is a worldwide problem with concomitant medical, social and economic burdens 1 for which pharmacotherapeutic approaches are limited 2 . Most patients with alcoholism will relapse within the first year of abstinence 3 , highlighting relapse as an important clinical issue. A main cause of relapse is cue-induced drug craving 4 , a process in which a cue that was previously associated with the reinforcing effects of alcohol elicits craving for alcohol itself, thereby increasing the likelihood of relapse. Thus, erasure of the memory for the cue-drug association is expected to reduce or prevent cue-induced relapse. Current conceptions of memory processes hold that after retrieval of a memory, it is reactivated and undergoes a process of destabilization followed by a process of reconsolidation. After destabilization, a temporary 'reconsolidation window' opens during which the memory becomes labile and can be strengthened or attenuated The mTORC1-mediated signaling pathway is required for the translation of a subset of dendritic proteins 12 and is implicated in synaptic plasticity 12,13 as well as memory processes 12 . Interestingly, mTORC1 has been reported to contribute to memory processes that are involved in cocaine-conditioned place preference and cue-induced reinstatement RESULTS Retrieval of alcohol-associated memories activates mTORC1 To determine whether the mTORC1 signaling pathway is activated after retrieval (reactivation) of alcohol-related memories (that is, during memory reconsolidation), we trained rats to voluntarily consume excessive amounts of alcohol in their home cage for 7 weeks using the intermittent access to 20% alcohol two bottle-choice procedur

Mass spectrometry identification of actin as a binding partner of RACK1.

<p><b><i>A</i></b> SH-SY5Y cells were treated with 10 μM FSK for 30 min, RACK1 was IPed from cell lysate and proteins were resolved by SDS-PAGE which was stained with Deep Purple™ to visualize proteins. Gel slices from both RACK1 and control IgG IPs were digested in-gel and the resulting peptides were submitted to tandem mass spectrometry (MS/MS) sequencing for protein identification, n = 2. <b><i>B</i></b> Identification of actin as a putative RACK1 binding protein. β-actin (ACTB), γ-actin (ACTG), alpha skeletal muscle actin (ACTS), aortic smooth muscle actin (ACTA), alpha cardiac muscle actin (ACTC) were identified in the RACK1 IP. A sum of score of peptides that were matched to the best hit within the family of homologous protein sequences (^<b>1</b>). Calculation of the percent sequence coverage included all peptides matched to the protein, i.e., both unique and common peptides (^<b>2</b>). Differentiation between ACTC, ACTS and ACTA proteins was not possible on the basis of the data since the identified peptides were common to all of them (^<b>3</b>). Differentiation between β-actin and γ-actin proteins was not possible on the basis of the data since the identified peptides were common to both isoforms (^<b>4</b>).</p

Disruption of actin filaments using cytochalasin D attenuates cAMP-mediated <i>BDNF</i> transcription without blocking the phosphorylation of CREB.

<p><b><i>A</i></b> SH-SY5Y cells were incubated with vehicle or 1 μM cytochalasin D for 15 min prior to treatment with 10 μM FSK for 1 h. <i>BDNF</i> and <i>GAPDH</i> mRNA levels were analyzed by RT-PCR. Histogram depicts the mean ratio of <i>BDNF</i> to <i>GAPDH</i> expressed as percent control (vehicle) ± SEM. Two-way ANOVA shows an interaction between FSK and cytochalasin D [P = 0.049]. ***p<0.001, *p<0.05, ns p = 0.177, Newman-Keuls post-hoc analysis. <b><i>B</i></b> SH-SY5Y cells were incubated with vehicle or 1 μM cytochalasin D for 15 min prior to treatment with 10 μM FSK for 30 min, then lysed in IP buffer. Proteins were resolved by SDS-PAGE and the phosphorylation of CREB on serine 133 was examined by western blot analysis. Histogram depicts the mean ratio of phospho-CREB to total CREB, expressed as percent control (vehicle) ± SEM. Two-way ANOVA shows an effect of FSK [P < 0.001] but no effect of cytochalasin D [P = 0.756] and no interaction [P = 0.966]. Subsequent analysis by the method of contrasts (two-tailed unpaired t-test) detected a significant difference between control and FSK in both vehicle and cytochalasin D groups. **p = 0.002 and ***p<0.001. A n = 9, B n = 3.</p