Christiane Pagès

Addictive and non-addictive drugs induce distinct and specific patterns of ERK activation in mouse brain

A major goal of research on addiction is to identify the molecular mechanisms of long‐lasting behavioural alterations induced by drugs of abuse. Cocaine and delta‐9‐tetrahydrocannabinol (THC) activate extracellular signal‐regulated kinase (ERK) in the striatum and blockade of the ERK pathway prevents establishment of conditioned place preference to these drugs. However, it is not known whether activation of ERK in the striatum is specific for these two drugs and/or this brain region. We studied the appearance of phospho‐ERK immunoreactive neurons in CD−1 mouse brain following acute administration of drugs commonly abused by humans, cocaine, morphine, nicotine and THC, or of other psychoactive compounds including caffeine, scopolamine, antidepressants and antipsychotics. Each drug generated a distinct regional pattern of ERK activation. All drugs of abuse increased ERK phosphorylation in nucleus accumbens, lateral bed nucleus of the stria terminalis, central amygdala and deep layers of prefrontal cortex, through a dopamine D1 receptor‐dependent mechanism. Although some non‐addictive drugs moderately activated ERK in a few of these areas, they never induced this combined pattern of strong activation. Antidepressants and caffeine activated ERK in hippocampus and cerebral cortex. Typical antipsychotics mildly activated ERK in dorsal striatum and superficial prefrontal cortex, whereas clozapine had no effect in the striatum, but more widespread effects in cortex and amygdala. Our results outline a subset of structures in which ERK activation might specifically contribute to the long‐term effects of drugs of abuse, and suggest mapping ERK activation in brain as a way to identify potential sites of action of psychoactive drugs.

Glutamate Induces Phosphorylation of Elk-1 and CREB, Along with c-fos Activation, via an Extracellular Signal-Regulated Kinase-Dependent Pathway in Brain Slices

ABSTRACT In cell culture systems, the TCF Elk-1 represents a convergence point for extracellular signal-related kinase (ERK) and c-Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK) subclasses of mitogen-activated protein kinase (MAPK) cascades. Its phosphorylation strongly potentiates its ability to activate transcription of the c-fos promoter through a ternary complex assembled on the c-fos serum response element. In rat brain postmitotic neurons, Elk-1 is strongly expressed (V. Sgambato, P. Vanhoutte, C. Pagès, M. Rogard, R. A. Hipskind, M. J. Besson, and J. Caboche, J. Neurosci. 18:214–226, 1998). However, its physiological role in these postmitotic neurons remains to be established. To investigate biochemically the signaling pathways targeting Elk-1 and c-fos in mature neurons, we used a semi-in vivo system composed of brain slices stimulated with the excitatory neurotransmitter glutamate. Glutamate treatment leads to a robust, progressive activation of the ERK and JNK/SAPK MAPK cascades. This corresponds kinetically to a significant increase in Ser383-phosphorylated Elk-1 and the appearance of c-fos mRNA. Glutamate also causes increased levels of Ser133-phosphorylated cyclic AMP-responsive element-binding protein (CREB) but only transiently relative to Elk-1 and c-fos. ERK and Elk-1 phosphorylation are blocked by the MAPK kinase inhibitor PD98059, indicating the primary role of the ERK cascade in mediating glutamate signaling to Elk-1 in the rat striatum in vivo. Glutamate-mediated CREB phosphorylation is also inhibited by PD98059 treatment. Interestingly, KN62, which interferes with calcium-calmodulin kinase (CaM-K) activity, leads to a reduction of glutamate-induced ERK activation and of CREB phosphorylation. These data indicate that ERK functions as a common component in two signaling pathways (ERK/Elk-1 and ERK/?/CREB) converging on the c-fospromoter in postmitotic neuronal cells and that CaM-Ks act as positive regulators of these pathways.

Parsing molecular and behavioral effects of cocaine in mitogen- and stress-activated protein kinase-1-deficient mice.

Although the induction of persistent behavioral alterations by drugs of abuse requires the regulation of gene transcription, the precise intracellular signaling pathways that are involved remain mainly unknown. Extracellular signal-regulated kinase (ERK) is critical for the expression of immediate-early genes in the striatum in response to cocaine and Δ9-tetrahydrocannabinol and for the rewarding properties of these drugs. Here we show that in mice a single injection of cocaine (10 mg/kg) activates mitogen- and stress-activated protein kinase 1 (MSK1) in dorsal striatum and nucleus accumbens. Cocaine-induced phosphorylation of MSK1 threonine 581 and cAMP response element-binding protein (CREB) serine 133 (Ser133) were blocked by SL327, a drug that prevents ERK activation. Cocaine increased the acetylation of histone H4 lysine 5 and phosphorylation of histone H3 Ser10, demonstrating the existence of drug-induced chromatin remodeling in vivo. In MSK1 knock-out (KO) mice CREB and H3 phosphorylation in response to cocaine (10 mg/kg) were blocked, and induction of c-Fos and dynorphin was prevented, whereas the induction of Egr-1 (early growth response-1)/zif268/Krox24 was unaltered. MSK1-KO mice had no obvious neurological defect but displayed a contrasted behavioral phenotype in response to cocaine. Acute effects of cocaine and dopamine D1 or D2 agonists were unaltered. Sensitivity to low doses, but not high doses, of cocaine was increased in the conditioned place preference paradigm, whereas locomotor sensitization to repeated injections of cocaine was decreased markedly. Our results show that MSK1 is a major striatal kinase, downstream from ERK, responsible for the phosphorylation of CREB and H3 and is required specifically for the induction of c-Fos and dynorphin as well as for locomotor sensitization.

Δ9‐tetrahydrocannabinol‐induced MAPK/ERK and Elk‐1 activation in vivo depends on dopaminergic transmission

It is now well established that central effects of Δ9‐tetrahydrocannabinol (THC), the main psychoactive component of marijuana, are mediated by CB1 cannabinoid receptors. However, intraneuronal signalling pathways activated in vivo by THC remain poorly understood. We show that acute administration of THC induces a progressive and transient activation (i.e. phosphorylation) of the mitogen activated protein kinase/extracellular signal‐regulated kinase (MAPK/ERK) in the dorsal striatum and the nucleus accumbens (NA). This activation, corresponding to both neuronal cell bodies and the surrounding neuropil, is totally inhibited by the selective antagonist of CB1 cannabinoid receptors, SR 141716A. However, blockade of dopaminergic (DA) D1 receptors by administration of SCH 23390, prior to THC, totally prevents ERK activation in the striatum, thus demonstrating a critical involvement of DA systems in THC‐induced ERK activation. DA‐D2 and glutamate receptors of NMDA subtypes also participate, albeit to a lesser extent, to THC‐induced ERK activation in the striatum, as shown after injection of selective antagonists (raclopride and MK801, respectively). Furthermore, THC‐induced phosphorylation of the transcription factor Elk‐1, and up‐regulation of zif268 mRNA expression are blocked by SL327, a specific inhibitor of MAPK/ERK kinase (MEK), the upstream kinase of ERK, as well as SCH 23390. Finally, using the place‐preference paradigm, we show that ERK inhibition blocks THC‐induced rewarding properties. Altogether, our data strongly support that ERK activation in the striatum is critically involved in long‐term neuronal adaptive responses underlying THC‐induced long‐term behaviours.

Cyclic Adenosine Monophosphate–Independent Tyrosine Phosphorylation of NR2B Mediates Cocaine-Induced Extracellular Signal-Regulated Kinase Activation

BACKGROUND Activation of the extracellular signal-regulated kinase (ERK) in the striatum is crucial for long-term behavioral alterations induced by drugs of abuse. In response to cocaine, ERK phosphorylation (i.e., activation) is restricted to medium-sized spiny neurons expressing dopamine D1 receptor (D1R) and depends on a concomitant stimulation of D1R and glutamate N-methyl-D-aspartate receptor (NMDAR). However, the mechanisms responsible for this activation, especially the respective contribution of D1R and NMDAR, remain unknown. METHODS We studied striatal neurons in culture stimulated with D1R agonist and/or glutamate and wild-type or genetically modified mice treated with cocaine. Biochemical, immunohistochemical, and imaging studies were performed. Mice were also subjected to behavioral experiments. RESULTS Stimulation of D1R cannot activate ERK by itself but potentiates glutamate-mediated calcium influx through NMDAR that is responsible for ERK activation. Potentiation of NMDAR by D1R depends on a cyclic adenosine monophosphate-independent signaling pathway, which involves tyrosine phosphorylation of the NR2B subunit of NMDAR by Src family kinases. We also demonstrate that the D1R/Src family kinases/NR2B pathway is responsible for ERK activation by cocaine in vivo. Inhibition of this pathway abrogates cocaine-induced locomotor sensitization and conditioned place preference. CONCLUSIONS Our results show that potentiation of NR2B-containing NMDAR by D1R is necessary and sufficient to trigger cocaine-induced ERK activation. They highlight a new cyclic adenosine monophosphate-independent pathway responsible for the integration of dopamine and glutamate signals by the ERK cascade in the striatum and for long-term behavioral alterations induced by cocaine.

Mitogen- and stress-activated protein kinase-1 deficiency is involved in expanded-huntingtin-induced transcriptional dysregulation and striatal death

Huntingtons disease (HD) is a neurodegenerative disorder due to an abnormal polyglutamine expansion in the N‐terminal region of huntingtin protein (Exp‐Htt). This expansion causes protein aggregation and neuronal dysfunction and death. Transcriptional dysregulation due to Exp‐Htt participates in neuronal death in HD. Here, using the R6/2 transgenic mouse model of HD, we identified a new molecular alteration that could account for gene dysregulation in these mice. Despite a nuclear activation of the mitogen‐activated protein kinase/extracellular regulated kinase (ERK) along with Elk‐1 and cAMP responsive element binding, two transcription factors involved in c‐Fos transcription, we failed to detect any histone H3 phosphorylation, which is expected after nuclear ERK activation. Accordingly, we found in the striatum of these mice a deficiency of mitogen‐ and stress‐activated kinase‐1 (MSK‐1), a kinase downstream ERK, critically involved in H3 phosphorylation and c‐Fos induction. We extended this observation to Exp‐Htt‐expressing striatal neurons and postmortem brains of HD patients. In vitro, knocking out MSK‐1 expression potentiated Exp‐Htt‐induced striatal death. Its overexpression induced H3 phosphorylation and c‐Fos expression and totally protected against striatal neurodegeneration induced by Exp‐Htt. We propose that MSK‐1 deficiency is involved in transcriptional dysregulation and striatal degeneration. Restoration of its expression and activity may be a new therapeutic target in HD. Roze E., Betuing, S., Deyts, C., Marcon, E., Brami‐Cherrier, K., Pages, C., Humbert, S., Mérienne, K., Caboche J. Mitogen‐ and stress‐activated protein kinase‐1 deficiency is involved in expanded‐hunting‐tin‐induced transcriptional dysregulation and striatal death. FASEB J. 22, 1083–1093 (2008)

Glutamate induces histone H3 phosphorylation but not acetylation in striatal neurons: role of mitogen- and stress-activated kinase-1

Chromatin remodelling is thought to play a key role in gene regulation that underlies long‐term synaptic plasticity and memory formation. The dynamic process of chromatin remodelling requires post‐translational modifications of histones, a group of highly basic proteins that are tightly linked to DNA. In the present study, we investigated histone H3 modifications in response to glutamate stimulation leading to c‐Fos and c‐Jun induction in an in vitro model system of striatal neurons in culture. Intracellular signalling pathways implicated in these modifications were analysed. Histone H3 acetylation was strong in basal conditions and unmodified by glutamate treatment. By contrast, glutamate induced a strong phosphorylation of histone H3 that was inhibited by selective inhibitors of the extracellular signal‐regulated kinase (ERK) and p38 mitogen‐activated protein kinase (p38 MAPK) pathways, U0126 and SB203580, respectively. Blocking activation of mitogen‐ and stress‐activated kinase 1 (MSK1), a kinase downstream ERK and p38 MAPK, by pharmacological approach or using striatal cells from MSK1 deficient mice, totally abolished H3 phosphorylation, as well as c‐Fos and c‐Jun induction. Chromatin immunoprecipitation assays confirmed increased levels of phosphorylated H3 at the c‐jun promoter. Altogether, our data highlight the crucial role of MSK1 in the nucleosomal response necessary for gene induction in neuronal cells.

INDUCIBILITY OF C-FOS PROTEIN IN VISUO-MOTOR SYSTEM AND LIMBIC STRUCTURES AFTER ACUTE AND REPEATED ADMINISTRATION OF NICOTINE IN THE RAT

To identify neuroanatomical substrates affected by nicotine, we have studied its effects after acute and repeated administration through the c‐Fos protein inducibility in various brain structures. Ninety minutes after acute nicotine (0.35 mg/kg, sc.) the number of c‐Fos‐like immunoreactive nuclei was consistently increased in visuo‐motor structures such as the superior colliculus, the medial terminal nucleus of accessory optic tract, and the nucleus of the optic tract. The anteroventral and lateroposterior thalamic nuclei, connected with the retina and involved in limbic processing, showed a c‐Fos induction. c‐Fos was preferentially induced in terminal fields of neurons of the ventral tegmental area such as the nucleus accumbens, the central amygdala, the lateral habenula, the lateral septum, as well as the cingulate, medial prefrontal, orbital and piriform cortices. In chronically treated rats (0.35 mg/kg sc., 3 x day for 14 days), the last nicotine injection given on the 15th day was still able to induce 90 minutes later c‐Fos protein in visuo‐motor, retino‐limbic, subcortical, and cortical limbic structures. Moreover, this chronic treatment produced an additional recruitment of c‐Fos‐positive nuclei in the cingulate cortex, the core and the ventral shell of the nucleus accumbens. c‐Fos induction after nicotine differs from that reported after other addictive drugs in terms of pattern and chronic inducibility, indicating that different mechanisms are involved for maintaining this transcription factor. In addition to a preferential sensitivity of mesolimbic dopaminergic neurons to nicotine, activation of visuo‐limbic and limbic regions could be relevant for understanding some context‐dependent and addictive behaviors produced by nicotine. Synapse 29:343–354, 1998.

Haloperidol protects striatal neurons from dysfunction induced by mutated huntingtin in vivo.

Huntingtons disease (HD) results from an abnormal polyglutamine extension in the N-terminal region of the huntingtin protein. This mutation causes preferential degeneration of striatal projection neurons. We previously demonstrated, in vitro, that dopaminergic D2 receptor stimulation acted synergistically with mutated huntingtin (expHtt) to increase aggregate formation and striatal death. In the present work, we extend these observations to an in vivo system based on lentiviral-mediated expression of expHtt in the rat striatum. The early and chronic treatment with the D2 antagonist haloperidol decanoate protects striatal neurons from expHtt-induced dysfunction, as analyzed by DARPP-32 and NeuN stainings. Haloperidol treatment also reduces aggregates formation, an effect that is maintained over time. These findings indicate that D2 receptors activation contributes to the deleterious effects of expHtt on striatal function and may represent an interesting early target to alter the subsequent course of neuropathology in HD.

Mitogen- and stress-activated protein kinase 1-induced neuroprotection in Huntington's disease: role on chromatin remodeling at the PGC-1-alpha promoter

Huntingtons disease (HD) is a neurodegenerative disorder due to abnormal polyglutamine expansion in huntingtin protein (Exp-Htt). This expansion causes protein aggregation, leading to neuronal dysfunction and death. We have previously shown that mitogen- and stress-activated kinase (MSK-1), a nuclear protein kinase involved in chromatin remodeling through histone H3 phosphorylation, is deficient in the striatum of HD patients and model mice. Restoring MSK-1 expression in cultured striatal cells prevented neuronal dysfunction and death induced by Exp-Htt. Here we extend these observations in a rat model of HD based on striatal lentiviral expression of Exp-Htt (LV-Exp-HTT). MSK-1 overexpression attenuated Exp-Htt-induced down-regulation of DARPP-32 expression 4 and 10 weeks after infection and enhanced NeuN staining after 10 weeks. LV-MSK-1 induced constitutive hyperphosphorylation of H3 and cAMP-responsive element binding protein (CREB), indicating that MSK-1 has spontaneous catalytic activity. MSK-1 overexpression also upregulated peroxisome proliferator-activated receptor γ coactivator alpha (PGC-1α), a transcriptional co-activator involved in mitochondrial biogenesis. Chromatin immunoprecipitation indicated that transcriptional regulation of PGC-1α is directly linked to increased binding of MSK-1, along with H3 and CREB phosphorylation of the PGC-1α promoter. MSK-1 knock-out mice showed spontaneous striatal atrophy as they aged, as well as higher susceptibility to systemic administration of the mitochondrial neurotoxin 3-NP. These results indicate that MSK-1 activation is an important and key event in the signaling cascade that regulates PGC-1α expression. Strategies aimed at restoring MSK-1 expression in the striatum might offer a new therapeutic approach to HD.