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Motor skills learning is classically associated with brain regions including cerebral and cerebellar cortices and basal ganglia nuclei. Less is known about the role of the hippocampus in the acquisition and storage of motor skills. Here, we show that mice receiving a long-term training in the accelerating rotarod display marked hippocampal transcriptional changes and reduced pyramidal neurons activity in the CA1 region when compared with naive mice. Then, we use mice in which neural ensembles are permanently labeled in an Egr1 activity-dependent fashion. Using these mice, we identify a subpopulation of Egr1-expressing pyramidal neurons in CA1 activated in short-term (STT) and long-term (LTT) trained mice in the rotarod task. When Egr1 is downregulated in the CA1 or these neuronal ensembles are depleted, motor learning is improved whereas their chemogenetic stimulation impairs motor learning performance. Thus, Egr1 organizes specific CA1 neuronal ensembles during the accelerating rotarod task that limit motor learning. These evidences highlight the role of the hippocampus in the control of this type of learning and we provide a possible underlying mechanism. SIGNIFICANCE STATEMENT It is a major topic in neurosciences the deciphering of the specific circuits underlying memory systems during the encoding of new information. However, the potential role of the hippocampus in the control of motor learning and the underlying mechanisms has been poorly addressed. In the present work we show how the hippocampus responds to motor learning and how the Egr1 molecule is one of the major responsible for such phenomenon controlling the rate of motor coordination performances.

International audience; DARPP-32 (dopamine-and cAMP-regulated phosphoprotein with an apparent Mr of 32 000), now also known as phosphoprotein phosphatase 1 regulatory subunit 1B (PPP1R1B), is a potent inhibitor of PP1 (also known as PPP1) when phosphorylated at Thr34 by cAMP-dependent protein kinase (PKA). DARPP-32 exhibits a remarkable regional distribution in brain, roughly similar to that of dopamine innervation. Its discovery was a culmination of the long-standing effort of Paul Greengard to understand the mechanisms through which neurotransmitters such as dopamine exert their effects on target neurons. DARPP-32 is particularly enriched in striatal projection neurons where it is regulated by numerous signals through which it integrates and amplifies responses to many stimuli. Molecular studies of DARPP-32 have revealed that its regulation and function are more complex than anticipated. It is phosphorylated on multiple sites by different protein kinases that modulate DARPP-32 properties. Primarily, when phosphorylated at Thr34 DARPP-32 is a potent inhibitor of PP1, whereas when phosphorylated at Thr75 by Cdk5 it inhibits PKA. Phosphorylation at serine residues by CK1 and CK2 modulates its intracellular localization and its sensitivity to kinases or phosphatases. Modeling studies provide evidence that the signaling pathways including DARPP-32 are endowed of strong robustness and bistable properties favoring switch-like responses. Thus DARPP-32 combined with a set of other distinct signaling molecules enriched in striatal projection neurons plays a key role in the characteristic properties and physiological function of these neurons.

Pyk2 is a non-receptor tyrosine kinase enriched in hippocampal neurons, which can be activated by calcium-dependent mechanisms. In neurons, Pyk2 is mostly localised in the cytosol and dendritic shafts but can translocate to spines and/or to the nucleus. Here, we explore the function of a new localisation of Pyk2 in mitochondria-associated membranes (MAMs), a subdomain of ER-mitochondria surface that acts as a signalling hub in calcium regulation. To test the role of Pyk2 in MAMs’ calcium transport, we used full Pyk2 knockout mice (Pyk2−/−) for in vivo and in vitro studies. Here we report that Pyk2−/− hippocampal neurons present increased ER-mitochondrial contacts along with defective calcium homeostasis. We also show how the absence of Pyk2 modulates mitochondrial dynamics and morphology. Taken all together, our results point out that Pyk2 could be highly relevant in the modulation of ER-mitochondria calcium efflux, affecting in turn mitochondrial function A.G. is a Ramón y Cajal fellow (RYC-2016¿19466) supported by a grant from Ministerio de Ciencia, Innovación y Universidades (RTI2018-094678-A-I00). RTI2018-094374-B-I00 to S.G., from Ministerio de Ciencia, Innovación y Universidades.

The persistent and experience-dependent nature of drug addiction may result in part from epigenetic alterations, including non-coding micro-RNAs (miRNAs), which are both critical for neuronal function and modulated by cocaine in the striatum. Two major striatal cell populations, the striato-nigral and striato-pallidal projection neurons, express, respectively, the D1 (D1-SPNs) and D2 (D2-SPNs) dopamine receptor, and display distinct but complementary functions in drug-evoked responses. However, a cell-type-specific role for miRNAs action has yet to be clarified. Here, we evaluated the expression of a subset of miRNAs proposed to modulate cocaine effects in the nucleus accumbens (NAc) and dorsal striatum (DS) upon sustained cocaine exposure in mice and showed that these selected miRNAs were preferentially upregulated in the NAc. We focused on miR-1 considering the important role of some of its predicted mRNA targets, Fosb and Npas4, in the effects of cocaine. We validated these targets in vitro and in vivo. We explored the potential of miR-1 to regulate cocaine-induced behavior by overexpressing it in specific striatal cell populations. In DS D1-SPNs miR-1 overexpression downregulated Fosb and Npas4 and reduced cocaine-induced CPP reinstatement, but increased cue-induced cocaine seeking. In DS D2-SPNs miR-1 overexpression reduced the motivation to self-administer cocaine. Our results indicate a role of miR1 and its target genes, Fosb and Npas4, in these behaviors and highlight a precise cell-type- and region-specific modulatory role of miR-1, illustrating the importance of cell-specific investigations. This work was supported by Fondation pour la Recherche Médicale (DEQ20150734352 to JC), Centre National pour la Recherche Scientifique (CNRS, B.F., A.G., V.K., P.P., P.V., J.C.), Institut National pour la Santé et la Recherche Médicale (INSERM; B.F., A.G., V.K., P.P., P.V., J.-A.G., J.C.), Sorbonne Université, Faculté des Sciences et Ingénierie (B.F., A.G., V.K., P.P., P.V., J.-A.G., J.C.) Labex Biopsy Investissements d’Avenir, ANR-11-IDEX-0004-02 (B.F., A.G., V.K., P.P., P.V., J.-A.G., J.C.), the Spanish Ministerio de Economía y Competitividad-MINECO (#SAF2017-84060-R-AEI/FEDER-UE; E.M.-G., L.D.-R., R.M.), the Spanish Instituto de Salud Carlos III, RETICS-RTA (#RD12/0028/0023; E.M.-G., L.D.-R., R.M.), the Generalitat de Catalunya, AGAUR (#2017-SGR-669; E.M.-G., L.D.-R., R.M.), ICREA-Acadèmia (#2015) and the Spanish Ministerio de Sanidad, Servicios Sociales e Igualdad, Plan Nacional Sobre Drogas (#PNSD-2017I068) to R.M., Fundació La Marató-TV3 (#2016/20-30) to E.M.-G. Fondation pour la Recherche Médicale (FRM#DPA20140629798) and ANR (ANR-16-CE16-0018) to J.A.G.

The most common treatment for patients with sickle cell disease (SCD) is the chemotherapeutic hydroxyurea, a therapy with pleiotropic effects, including increasing fetal hemoglobin (HbF) in red blood cells and reducing adhesion of white blood cells to the vascular endothelium. Hydroxyurea has been proposed to mediate these effects through a mechanism of increasing cellular cGMP levels. An alternative path to increasing cGMP levels in these cells is through the use of phosphodiesterase-9 inhibitors that selectively inhibit cGMP hydrolysis and increase cellular cGMP levels. We have developed a novel, potent and selective phosphodiesterase-9 inhibitor (IMR-687) specifically for the treatment of SCD. IMR-687 increased cGMP and HbF in erythroid K562 and UT-7 cells and increased the percentage of HbF positive erythroid cells generated in vitro using a two-phase liquid culture of CD34+ progenitors from sickle cell blood or bone marrow. Oral daily dosing of IMR-687 in the Townes transgenic mouse SCD model, increased HbF and reduced red blood cell sickling, immune cell activation and microvascular stasis. The IMR-687 reduction in red blood cell sickling and immune cell activation was greater than that seen with physiological doses of hydroxyurea. In contrast to other described phosphodiesterase-9 inhibitors, IMR-687 did not accumulate in the central nervous system, where it would inhibit phosphodiesterase-9 in neurons, or alter rodent behavior. IMR-687 was not genotoxic or myelotoxic and did not impact fertility or fetal development in rodents. These data suggest that IMR-687 may offer a safe and effective oral alternative for hydroxyurea in the treatment of SCD.

Food intake is tightly regulated by homeostatic and reward mechanisms and the adequate function of both is necessary for the proper maintenance of energy balance. Ghrelin impacts on these two levels to induce feeding. In this review, we present the actions of ghrelin in food reward, including their dependence on other relevant modulators implicated in the motivational aspects of feeding, including dopamine, opioid peptides, and endocannabinoids. We also describe the interaction between brain areas involved in homeostatic regulation of feeding and the reward system, with a special emphasis on the role of arcuate nucleus melanocortins and lateral hypothalamus orexins in ghrelin function. Finally, we briefly discuss the actions of ghrelin in food reward in obesity. We propose that new insights into the mechanism of action of ghrelin in the rewarding and motivational control of food intake will help to understand food-related disorders including obesity and anorexia.

D1 dopamine receptors play an important role in the effects of cocaine. Here we investigated the role of neurons which express these receptors (D1-neurons) in the acute locomotor effects of cocaine and the locomotor sensitization observed after a second injection of this drug. We inhibited D1-neurons using double transgenic mice conditionally expressing the inhibitory Gi-coupled designer receptor exclusively activated by designer drugs (Gi-DREADD) in D1-neurons. Chemogenetic inhibition of D1-neurons by a low dose of clozapine (0.1 mg/kg) decreased the induction of Fos in striatal neurons. It diminished the basal locomotor activity and acute hyper-locomotion induced by cocaine (20 mg/kg). Clozapine 0.1 mg/kg had no effect by itself and did not alter cocaine effects in non-transgenic mice. Inhibition of D1-neurons during the first cocaine administration reduced the sensitization of the locomotor response in response to a second cocaine administration ten days later. At day 11, inhibition of D1-neurons by clozapine stimulation of Gi-DREADD, prevented the expression of the sensitized locomotor response, whereas at day 12, in the absence of clozapine and D1-neurons inhibition, all mice displayed a sensitized response to cocaine. These results show that chemogenetic inhibition of D1-neurons decreases spontaneous and cocaine-induced locomotor activity. It blunts the induction and prevents the expression of sensitization in a two-injection protocol of sensitization but does not alter established sensitization. Our study further supports the central role of D1-neurons in mediating the acute locomotor effects of cocaine and its sensitization.

International audience; Abstract The striatum is critical for cocaine-induced locomotor responses. Although the role of D1 receptor-expressing neurons is established, underlying molecular pathways are not fully understood. We studied the role of Pyk2, a non-receptor, calcium-dependent protein-tyrosine kinase. The locomotor coordination and basal activity of Pyk2 knock-out mice were not altered and major striatal protein markers were normal. Cocaine injection increased Pyk2 tyrosine phosphorylation in mouse striatum. Pyk2-deficient mice displayed decreased locomotor response to acute cocaine injection. In contrast, locomotor sensitization and conditioned place preference were normal. Cocaine-activated ERK phosphorylation, a signaling pathway essential for these late responses, was unaltered. Conditional deletion of Pyk2 in the nucleus accumbens or in D1 neurons reproduced decreased locomotor response to cocaine, whereas deletion of Pyk2 in the dorsal striatum or in A 2A receptor-expressing neurons did not. In mice lacking Pyk2 in D1-neurons locomotor response to D1 agonist SKF-81297, but not to an anticholinergic drug, was blunted. Our results identify Pyk2 as a regulator of acute locomotor responses to psychostimulants. They highlight the role of tyrosine phosphorylation pathways in striatal neurons and suggest that changes in Pyk2 expression or activation may alter specific responses to drugs of abuse, or possibly other behavioral responses linked to dopamine action.

Permanent tagging of neuronal ensembles activated in specific experimental situations is an important objective to study their properties and adaptations. In the context of learning and memory, these neurons are referred to as engram neurons. Here, we describe and characterize a novel mouse line, Egr1-CreER

International audience; Depression is a leading cause of disability worldwide. Circadian abnormalities and mood changes are symptoms of depression. The psychostimulant caffeine alters wakefulness and alleviates other depression-related symptoms during chronic intake, but the underlying mechanisms are unclear. It is not known, whether and how acute caffeine administration affects mood. Molecular approaches, transgenic mouse models, pharmacological intervention and behavioral analysis were combined to uncover a regulatory pathway, which connects caffeine action with diurnal signaling via the key dopaminergic protein DARPP-32 and alters mood-related phenotypes in mice, which are often assessed in the context of antidepressant action. We observed that Thr75-DARPP-32 binds to the circadian regulator CLOCK and disrupts CLOCK:BMAL1 chromatin binding, thereby affecting gene expression. T75A-DARPP-32 mutant mice show reduced caffeine effects on CLOCK:BMAL1 and lack caffeine-induced effects on mood. This study provides a link between caffeine, diurnal signaling and mood-related behaviors, which may open new perspectives for our understanding of antidepressant mechanisms in the mouse brain.

Although abnormal structural and functional connectivity in the striatum during neurological disorders has been reported using functional magnetic resonance imaging (fMRI), the effects of cell-type specific neuronal stimulation on fMRI and related behavioral alterations are not well understood. In this study, we successfully combined DREADD-technology with fMRI (chemo-fMRI) to investigate the alterations of spontaneous neuronal activity induced by the unilateral activation of dopamine D1 receptor-expressing neurons (D1-neurons) in the mouse dorsal striatum (DS). We compared the effects of two different DREADD ligands, clozapine (CLZ) and clozapine-N-oxide (CNO), on behavior and fMRI. We found that the effects of CLZ on behavior were more rapid than those of CNO. In fMRI, both systemic CLZ and CNO administrations, which evoked unilateral activations of D1-neurons in DS, increased the fractional amplitude of low frequency fluctuations (fALFF) in the thalamus and bilateral cortex. In addition, we found the increased gamma-band of local field potentials in DS and bilateral cortex after CLZ-evoked unilateral activation of D1-neuron in the striatum. These results provide bases for better interpretation of cell type-specific activity changes in fMRI.

Action control is a key brain function determining the survival of animals in their environment. In mammals, neurons expressing dopamine D2 receptors (D2R) in the dorsal striatum (DS) and the nucleus accumbens (Acb) jointly but differentially contribute to the fine regulation of movement. However, their region-specific molecular features are presently unknown. By combining RNAseq of striatal D2R neurons and histological analyses, we identified hundreds of novel region-specific molecular markers, which may serve as tools to target selective subpopulations. As a proof of concept, we characterized the molecular identity of a subcircuit defined by WFS1 neurons and evaluated multiple behavioral tasks after its temporally-controlled deletion of D2R. Consequently, conditional D2R knockout mice displayed a significant reduction in digging behavior and an exacerbated hyperlocomotor response to amphetamine. Thus, targeted molecular analyses reveal an unforeseen heterogeneity in D2R-expressing striatal neuronal populations, underlying specific D2R’s functional features in the control of specific motor behaviors., Striatal dopaminoceptive neurons are molecularly and functionally heterogeneous. Here, the authors provide the translatome of D2R striatal neurons and identified hundreds of region specific molecular markers as well as testing their resource to shed light into function.

International audience; Macrophage infiltration in mammary tumors is associated with enhanced tumor progression, metastasis, and poor clinical outcome, and considered as target for therapeutic intervention. By using different genetic mouse models, the authors show that ablation of the tyrosine kinase PYK2, either in breast cancer cells, only in the tumor microenvironment, or in both, markedly reduces the number of infiltrating tumor macrophages and concomitantly inhibits tumor angiogenesis and tumor growth. Strikingly, PYK2 ablation only in macrophages is sufficient to induce similar effects. These phenotypic changes are associated with reduced monocyte recruitment and a substantial decrease in tumor-associated macrophages (TAMs). Mechanistically, the authors show that PYK2 mediates mutual communication between breast cancer cells and macrophages through critical effects on key receptor signaling. Specifically, PYK2 ablation inhibits Notch1 signaling and consequently reduces CCL2 secretion by breast cancer cells, and concurrently reduces the levels of CCR2, CXCR4, IL-4R , and Stat6 activation in macrophages. These bidirectional effects modulate monocyte recruitment, macrophage polarization, and tumor angiogenesis. The expression of PYK2 is correlated with infiltrated macrophages in breast cancer patients, and its effects on macrophage infiltration and pro-tumorigenic phenotype suggest that PYK2 targeting can be utilized as an effective strategy to modulate TAMs and possibly sensitize breast cancer to immunotherapy.

[Background] Depression is the most common psychiatric condition in Huntington’s disease (HD), with rates more than twice those found in the general population. At the present time, there is no established molecular evidence to use as a basis for depression treatment in HD. Indeed, in some patients, classic antidepressant drugs exacerbate chorea or anxiety. Cyclin-dependent kinase 5 (Cdk5) has been involved in processes associated with anxiety and depression. This study evaluated the involvement of Cdk5 in the development and prevalence of depressive-like behaviors in HD and aimed to validate Cdk5 as a target for depression treatment. [Methods] We evaluated the impact of pharmacological inhibition of Cdk5 in depressive-like and anxiety-like behaviors in Hdh+/Q111 knock-in mutant mice by using a battery of behavioral tests. Biochemical and morphological studies were performed to define the molecular mechanisms acting downstream of Cdk5 activation. A double huntingtin/DARPP-32 (dopamine- and cAMP-regulated phosphoprotein 32) knock-in mutant mouse was generated to analyze the role of DARPP-32 in HD depression. [Results] We found that Hdh+/Q111 mutant mice exhibited depressive-like, but not anxiety-like, behaviors starting at 2 months of age. Cdk5 inhibition by roscovitine infusion prevented depressive-like behavior and reduced DARPP-32 phosphorylation at Thr75 in the nucleus accumbens. Hdh+/Q111 mice heterozygous for DARPP-32 Thr75Ala point mutation were resistant to depressive-like behaviors. We identified β-adducin phosphorylation as a Cdk5 downstream mechanism potentially mediating structural spine plasticity changes in the nucleus accumbens and depressive-like behavior. [Conclusions] These results point to Cdk5 in the nucleus accumbens as a critical contributor to depressive-like behaviors in HD mice by altering DARPP-32/β-adducin signaling and disrupting the dendritic spine cytoskeleton., This work was supported by the Ministerio de Economía y Competitividad (Grant No. SAF2015-67474-R;MINECO/FEDER [to SG] and Grant No. SAF2017-88076-R [to JA]), Centro de Investigaciones Biomédicas en Red sobre Enfermedades Neurodegenerativas (Grant No. CB06/05/0054 [to JA]), Agencia de Gestión de Ayudas Universitarias y de Investigación (Grant No. 2017SGR717 [to AC]). and Ramon y Cajal fellowship (Grant No. RYC-2016-19466 [to AG]). J-AG’s laboratory was supported in part by Inserm, the Université Pierre et Marie Curie (Sorbonne Université), the Fondation pour la Recherche Médicale, and the Laboratory of Excellence of Biology for Psychiatry.

It has been well documented that neurotrophins, including brain-derived neurotrophic factor (BDNF), are severely affected in Alzheimer's disease (AD), but their administration faces a myriad of technical challenges. Here we took advantage of the early astrogliosis observed in an amyloid mouse model of AD (5xFAD) and used it as an internal sensor to administer BDNF conditionally and locally. We first demonstrate the relevance of BDNF release from astrocytes by evaluating the effects of coculturing WT neurons and BDNF-deficient astrocytes. Next, we crossed 5xFAD mice with pGFAP:BDNF mice (only males were used) to create 5xFAD mice that overexpress BDNF when and where astrogliosis is initiated (5xF:pGB mice). We evaluated the behavioral phenotype of these mice. We first found that BDNF from astrocytes is crucial for dendrite outgrowth and spine number in cultured WT neurons. Double-mutant 5xF:pGB mice displayed improvements in cognitive tasks compared with 5xFAD littermates. In these mice, there was a rescue of BDNF/TrkB downstream signaling activity associated with an improvement of dendritic spine density and morphology. Clusters of synaptic markers, PSD-95 and synaptophysin, were also recovered in 5xF:pGB compared with 5xFAD mice as well as the number of presynaptic vesicles at excitatory synapses. Additionally, experimentally evoked LTP in vivo was increased in 5xF:pGB mice. The beneficial effects of conditional BDNF production and local delivery at the location of active neuropathology highlight the potential to use endogenous biomarkers with early onset, such as astrogliosis, as regulators of neurotrophic therapy in AD. SIGNIFICANCE STATEMENT Recent evidence places astrocytes as pivotal players during synaptic plasticity and memory processes. In the present work, we first provide evidence that astrocytes are essential for neuronal morphology via BDNF release. We then crossed transgenic mice (5xFAD mice) with the transgenic pGFAP-BDNF mice, which express BDNF under the GFAP promoter. The resultant double-mutant mice 5xF:pGB mice displayed a full rescue of hippocampal BDNF loss and related signaling compared with 5xFAD mice and a significant and specific improvement in all the evaluated cognitive tasks. These improvements did not correlate with amelioration of β amyloid load or hippocampal adult neurogenesis rate but were accompanied by a dramatic recovery of structural and functional synaptic plasticity.

Parkinson's disease (PD) is characterized by severe locomotor deficits due to the disappearance of dopamine (DA) from the dorsal striatum. The development of PD symptoms and treatment-related complications such as dyskinesia have been proposed to result from complex alterations in intracellular signaling in both direct and indirect pathway striatal projection neurons (dSPNs and iSPNs, respectively) following loss of DA afferents. To identify cell-specific and dynamical modifications of signaling pathways associated with PD, we used a hemiparkinsonian mouse model with 6-hydroxydopamine (6-OHDA) lesion combined with two-photon fluorescence biosensors imaging in adult corticostriatal slices. After DA lesion, extracellular signal-regulated kinase (ERK) activation was increased in response to DA D1 receptor (D1R) or α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) stimulation. The cAMP-dependent protein kinase (PKA) pathway contributing to ERK activation displayed supersensitive responses to D1R stimulation after 6-OHDA lesion. This cAMP/PKA supersensitivity was specific of D1R-responding SPNs and resulted from Gα

On behalf of the IMAGEN consortium., [Background]: The mesolimbic dopamine system, composed primarily of dopaminergic neurons in the ventral tegmental area that project to striatal structures, is considered to be the key mediator of reinforcement-related mechanisms in the brain. Prompted by a genome-wide association meta-analysis implicating the Ras-specific guanine nucleotide-releasing factor 2 (RASGRF2) gene in the regulation of alcohol intake in men, we have recently shown that male Rasgrf2–/– mice exhibit reduced ethanol intake and preference accompanied by a perturbed mesolimbic dopamine system. We therefore propose that these mice represent a valid model to further elucidate the precise genes and mechanisms regulating mesolimbic dopamine functioning. [Methods]: Transcriptomic data from the nucleus accumbens (NAcc) of male Rasgrf2–/– mice and wild-type controls were analyzed by weighted gene coexpression network analysis (WGCNA). We performed follow-up genetic association tests in humans using a sample of male adolescents from the IMAGEN study characterized for binge drinking (n = 905) and ventral striatal activation during an fMRI reward task (n = 608). [Results]: The WGCNA analyses using accumbal transcriptomic data revealed 37 distinct “modules,” or functionally related groups of genes. Two of these modules were significantly associated with Rasgrf2 knockout status: M5 (p < 0.001) and M6 (p < 0.001). In follow-up translational analyses we found that human orthologues for the M5 module were significantly (p < 0.01) enriched with genetic association signals for binge drinking in male adolescents. Furthermore, the most significant locus, originating from the EH-domain containing 4 (EHD4) gene (p < 0.001), was also significantly associated with altered ventral striatal activity in male adolescents performing an fMRI reward task (pempirical < 0.001). [Limitations]: It was not possible to determine the extent to which the M5 module was dysregulated in Rasgrf2–/– mice by perturbed mesolimbic dopamine signalling or by the loss of Rasgrf2 function in the NAcc. [Conclusion]: Taken together, our findings indicate that the accumbal M5 module, initially identified as being dysregulated in male Rasgrf2–/– mice, is also relevant for human alcohol-related phenotypes potentially through the modulation of reinforcement mechanisms in the NAcc. We therefore propose that the genes comprising this module represent important candidates for further elucidation within the context of alcohol-related phenotypes., This work was supported by the European Union-funded FP6 Integrated Project IMAGEN (reinforcement-­related behaviour in normal brain function and psychopathology) (LSHM-CT2007-037286), the FP7 projects IMAGEMEND, MATRICS and the Innovative Medicine Initiative Project EU-AIMS (115300-2), as well as the Medical Research Council Programme grant “Developmental pathways into adolescent substance abuse” (93558). Further support was provided by the Swedish Funding Agency FORMAS, the German Bundesministerium für Bildung und Forschung (NGFN Plus; FKZ: 01GS08152 and eMED „Alcoholism“) and the Deutsche Forschungsgemeinschaft (DFG): Reinhart-Koselleck Award SP 383/5-1, European Research Council and FP7 SynSys (to JAG) and Ecole des Neurosciences de Paris-Ile de France (ENP) and City of Paris (to MM), Grants PS09/01979 and RD06/0020/0000 from MINECO, Spain (to ES).

International audience; Microglial cells are resident innate immune cells of the brain that constantly scan their environment with their long processes and, upon disruption of homeostasis, undergo rapid morphological changes. For example, a laser lesion induces in a few minutes an oriented growth of microglial processes, also called "directional motility", toward the site of injury. A similar effect can be obtained by delivering locally ATP or serotonin (5-hydroxytryptamine [5-HT]). In this article, we describe a protocol to induce a directional growth of microglial processes toward a local application of ATP or 5-HT in acute brain slices of young and adult mice and to image this attraction over time by multiphoton microscopy. A simple method of quantification with free and open-source image analysis software is proposed. A challenge that still characterizes acute brain slices is the limited time, decreasing with age, during which the cells remain in a physiological state. This protocol, thus, highlights some technical improvements (medium, air-liquid interface chamber, imaging chamber with a double perfusion) aimed at optimizing the viability of microglial cells over several hours, especially in slices from adult mice.

Major depressive disorder (MDD) is a common disorder with a variety of symptoms including mood alterations, anhedonia, sleep and appetite disorders, and cognitive disturbances. Stressful life events are among the strongest risk factors for developing MDD. At the cellular level, chronic stress results in the modification of dendritic spine morphology and density. Here, we study the role of Pyk2 in the development of depressive-like symptoms induced by a model of chronic unpredictable mild stress (CUMS). Pyk2 is a non-receptor calcium-dependent protein-tyrosine kinase highly expressed in the forebrain principal neurons and involved in spine structure and density regulation. We show that Pyk2 knockout mice are less affected to anxiety-like and anhedonia-like phenotypes induced by the CUMS paradigm. Using region-specific knockout, we demonstrate that this phenotype is fully recapitulated by selective Pyk2 inactivation in the amygdala. We also show that in the absence of Pyk2 the spine alterations, PSD-95 clustering, and NMDA receptors changes induced by the CUMS paradigm are prevented. Our results reveal a possible role for Pyk2 in the response to stress and in synaptic markers expression and spine density regulation in the amygdala. We suggest that Pyk2 contributes to stress-induced responses through micro-structural changes and that its deficit may contribute to the resilience to chronic stress A.G. is a Ramón y Cajal fellow (RYC-2016-19466). This work was supported by the NARSAD foundation (young investigator grant 2016 (Ref. 24803) to A.G.) and Ministerio de Ciencia e Innovació n (SAF2015-67474-R; MINECO/FEDER to S.G.). J.-A.G. lab was supported in part by Inserm, the Université Pierre et Marie Curie (UPMC, Sorbonne Université), the Fondation pour la Recherche Médicale, and the Bio-Psy (Biology for Psychiatry) laboratory of excellence. O.A.-M. was funded by the ISCIII/SERGAS thought a research contract “Sara Borrell” (CD14/ 00091). JA lab was supported by the Spanish Ministry of Economy and Competitiveness (MINECO) (SAF-2014-57160R, SAF-2017-88076-R), Fundació Marató TV3 to J.A. and Instituto Carlos III: Centro de Investigación Biomédica en Red sobre enfermedades neurodegenerativas (CIBERNED), and RETICS RD12/ 0019/0002 to J.A.) SI

International audience; In retinal detachment (RD), photoreceptor death and permanent vision loss are caused by neurosensory retina separating from the retinal pigment epithelium because of subretinal fluid (SRF), and successful surgical reattachment is not predictive of total visual recovery. As retinal iron overload exacerbates cell death in retinal diseases, we assessed iron as a predictive marker and therapeutic target for RD. In the vitreous and SRF from patients with RD, we measured increased iron and transferrin (TF) saturation that is correlated with poor visual recovery. In ex vivo and in vivo RD models, iron induces immediate necrosis and delayed apoptosis. We demonstrate that TF decreases both apoptosis and necroptosis induced by RD, and using RNA sequencing, pathways mediating the neuroprotective effects of TF are identified. Since toxic iron accumulates in RD, we propose TF supplementation as an adjunctive therapy to surgery for improving the visual outcomes of patients with RD.

Overgeneralization of conditioned threat responses is a robust clinical marker of anxiety disorders. In overgeneralization, responses that are appropriate to threat-predicting cues are evoked by perceptually similar safety-predicting cues. Inappropriate learning of conditioned threat responses may thus form an etiological basis for anxiety disorders. The role of dopamine (DA) in memory encoding is well established. Indeed by signaling salience and valence, DA is thought to facilitate discriminative learning between stimuli representing safety or threat. However, the neuroanatomical and biochemical substrates through which DA modulates overgeneralization of threat responses remain poorly understood. Here we report that the modulation of DA D2 receptor (D2R) signaling bidirectionally regulates the consolidation of fear responses. While the blockade of D2R induces generalized threat responses, its stimulation facilitates discriminative learning between stimuli representing safety or threat. Moreover, we show that controlled threat generalization requires the coordinated activation of D2R in the bed nucleus of the stria terminalis and the central amygdala. Finally, we identify the mTORC1 cascade activation as an important molecular event by which D2R mediates its effects. These data reveal that D2R signaling in the extended amygdala constitutes an important checkpoint through which DA participates in the control of threat processing and the emergence of overgeneralized threat responses.Molecular Psychiatry advance online publication, 19 January 2016; doi:10.1038/mp.2015.210.

International audience; The rise in the prevalence of neurodegenerative diseases parallels the rapid increase in human lifespan. Despite intensive research, the molecular and cellular mechanisms underlying the onset and progression of these devastating diseases with age are still poorly understood. Many aspects of these diseases have been modelled successfully in experimental animals such as the mouse, the zebrafish Brachydanio rero, the nematode worm Caenorhaditis elegans and the fruit fly Drosophila melanogaster. This review will focus on the advantages offered by the genetic tools available in Drosophila for combining powerful strategies in order to tackle the causative factors of these complex pathologies and help to elaborate efficient drugs to treat them.

Althoughl-3,4-dihydroxyphenylalanine (l-DOPA) remains the reference treatment of Parkinson's disease, its long-term beneficial effects are hindered byl-DOPA-induced dyskinesia (LID). In the dopamine (DA)-denervated striatum,l-DOPA activates DA D1receptor (D1R) signaling, including cAMP-dependent protein kinase A (PKA) and extracellular signal-regulated kinase (ERK), two responses associated with LID. However, the cause of PKA and ERK activation, their respective contribution to LID, and their relationship are not known. In striatal neurons, D1R activates adenylyl-cyclase through Gαolf, a protein upregulated after lesion of DA neurons in rats and in patients. We report here that increased Gαolflevels in hemiparkinsonian mice are correlated with LID after chronicl-DOPA treatment. To determine the role of this upregulation, we performed unilateral lesion in mice lacking one allele of theGnalgene coding for Gαolf(Gnal+/−). Despite an increase in the lesioned striatum, Gαolflevels remained below those of unlesioned wild-type mice. InGnal+/−mice, the lesion-inducedl-DOPA stimulation of cAMP/PKA-mediated phosphorylation of GluA1 Ser845 and DARPP-32 (32 kDa DA- and cAMP-regulated phosphoprotein) Thr34 was dramatically reduced, whereas ERK activation was preserved. LID occurrence was similar inGnal+/+andGnal+/−mice after a 10-dl-DOPA (20 mg/kg) treatment. Thus, in lesioned animals, Gαolfupregulation is critical for the activation byl-DOPA of D1R-stimulated cAMP/PKA but not ERK signaling. Although the cAMP/PKA pathway appears to be required for LID development, our results indicate that its activation is unlikely to be the main source of LID. In contrast, the persistence ofl-DOPA-induced ERK activation inGnal+/−mice supports its causal role in LID development.

Conditioned place preference (CPP) is widely used for evaluating the rewarding effects of drugs. Like other memories, CPP is proposed to undergo reconsolidation during which it is unstable and sensitive to pharmacological inhibition. Previous studies have shown that cocaine CPP can be apparently erased by extracellular signal-regulated kinase (ERK) pathway inhibition during cocaine reconditioning (re-exposure to the drug-paired environment in the presence of the drug). Here, we show that blockade of D1 receptors during reconditioning prevented ERK activation and induced a loss of CPP. However, we also unexpectedly observed a CPP disappearance in mice that underwent testing and reconditioning with cocaine alone, specifically in strong conditioning conditions. The loss was due to the intermediate test. CPP was not recovered with reconditioning or priming in the short term, but it spontaneously reappeared after a month. When we challenged the D1 antagonist-mediated erasure, we observed that both a high dose of cocaine and a first CPP test were required for this effect. Our results also suggest a balance between D1-dependent ERK pathway activation and an A2a-dependent mechanism in D2 striatal neurons in controlling CPP expression. Our data reveal that, paradoxically, a simple CPP test can induce a complete (but transient) loss of place preference following strong but not weak cocaine conditioning. This study emphasizes the complex nature of CPP memory and the importance of multiple parameters that must be taken into consideration when investigating reconsolidation.

Pyk2 is a Ca2+-activated non-receptor tyrosine kinase enriched in forebrain neurons and involved in synaptic regulation. Human genetic studies associated PTK2B, the gene coding Pyk2, with risk for Alzheimer's disease (AD). We previously showed that Pyk2 is important for hippocampal function, plasticity, and spine structure. However, its potential role in AD is unknown. To address this question we used human brain samples and 5XFAD mice, an amyloid mouse model of AD expressing mutated human amyloid precursor protein and presenilin1. In the hippocampus of 5XFAD mice and in human AD patients' cortex and hippocampus, Pyk2 total levels were normal. However, Pyk2 Tyr-402 phosphorylation levels, reflecting its autophosphorylation-dependent activity, were reduced in 5XFAD mice at 8 months of age but not 3 months. We crossed these mice with Pyk2−/− mice to generate 5XFAD animals devoid of Pyk2. At 8 months the phenotype of 5XFAD x Pyk2−/− double mutant mice was not different from that of 5XFAD. In contrast, overexpression of Pyk2 in the hippocampus of 5XFAD mice, using adeno-associated virus, rescued autophosphorylated Pyk2 levels and improved synaptic markers and performance in several behavioral tasks. Both Pyk2−/− and 5XFAD mice showed an increase of potentially neurotoxic Src cleavage product, which was rescued by Pyk2 overexpression. Manipulating Pyk2 levels had only minor effects on Aβ plaques, which were slightly decreased in hippocampus CA3 region of double mutant mice and increased following overexpression. Our results show that Pyk2 is not essential for the pathogenic effects of human amyloidogenic mutations in the 5XFAD mouse model. However, the slight decrease in plaque number observed in these mice in the absence of Pyk2 and their increase following Pyk2 overexpression suggest a contribution of this kinase in plaque formation. Importantly, a decreased function of Pyk2 was observed in 5XFAD mice, indicated by its decreased autophosphorylation and associated Src alterations. Overcoming this deficit by Pyk2 overexpression improved the behavioral and molecular phenotype of 5XFAD mice. Thus, our results in a mouse model of AD suggest that Pyk2 impairment may play a role in the symptoms of the disease.

In Parkinson's disease, long-term dopamine replacement therapy is complicated by the appearance ofl-DOPA-induced dyskinesia (LID). One major hypothesis is that LID results from an aberrant transcriptional program in striatal neurons induced byl-DOPA and triggered by the activation of ERK. To identify these genes, we performed transcriptome analyses in the striatum in 6-hydroxydopamine-lesioned mice. A time course analysis (0–6 h after treatment withl-DOPA) identified an acute signature of 709 genes, among which genes involved in protein phosphatase activity were overrepresented, suggesting a negative feedback on ERK activation byl-DOPA.l-DOPA-dependent deregulation of 28 genes was blocked by pretreatment with SL327, an inhibitor of ERK activation, and 26 genes were found differentially expressed between highly and weakly dyskinetic animals after treatment withl-DOPA. The intersection list identified five genes:FosB,Th,Nptx2,Nedd4l, andCcrn4l. Nptx2encodes neuronal pentraxin II (or neuronal activity-regulated pentraxin, Narp), which is involved in the clustering of glutamate receptors. We confirmed increasedNptx2expression afterl-DOPA and its blockade by SL327 using quantitative RT-PCR in independent experiments. Using an escalatingl-DOPA dose protocol, LID severity was decreased in Narp knock-out mice compared with their wild-type littermates or after overexpression of a dominant-negative form of Narp in the striatum. In conclusion, we have identified a molecular signature induced byl-DOPA in the dopamine-denervated striatum that is dependent on ERK and associated with LID. Here, we demonstrate the implication of one of these genes,Nptx2, in the development of LID.

Dystonia is a movement disorder characterized by sustained or intermittent muscle contractions and its pathophysiological mechanisms are still poorly understood. Dominant mutations of the GNAL gene are a cause of isolated dystonia (DYT25) in patients. Some mutations result in a complete loss of function of the encoded protein, Gα(olf), an adenylyl-cyclase-stimulatory G-protein highly enriched in striatal projection neurons, where it mediates the actions of dopamine and adenosine. We used male and female heterozygous Gnal knock-out mice (Gnal(+/−)) to study how GNAL haplodeficiency is implicated in dystonia. In basal conditions, no overt dystonic movements or postures or change in locomotor activity were observed. However, Gnal haploinsufficiency altered self-grooming, motor coordination, and apparent motivation in operant conditioning, as well as spine morphology and phospho-CaMKIIβ in the striatum. After systemic administration of oxotremorine, an unselective cholinergic agonist, Gnal(+/−) mice developed more abnormal postures and movements than WT mice. These effects were not caused by seizures as indicated by EEG recordings. They were prevented by the M1-preferring muscarinic antagonists, telenzepine, pirenzepine, and trihexyphenidyl, which alleviate dystonic symptoms in patients. The motor defects were worsened by mecamylamine, a selective nicotinic antagonist. These oxotremorine-induced abnormalities in Gnal(+/−) mice were replicated by oxotremorine infusion into the striatum, but not into the cerebellum, indicating that defects in striatal neurons favor the appearance of dystonia-like movement alterations after oxotremorine. Untreated and oxotremorine-treated Gnal(+/−) mice provide a model of presymptomic and symptomatic stages of DYT25-associated dystonia, respectively, and clues about the mechanisms underlying dystonia pathogenesis. SIGNIFICANCE STATEMENT Adult-onset dystonia DYT25 is caused by dominant loss-of-function mutations of GNAL, a gene encoding the stimulatory G-protein Gαolf, which is critical for activation of the cAMP pathway in the striatal projection neurons. Here, we demonstrate that Gnal-haplodeficient mice have a mild neurological phenotype and display vulnerability to developing dystonic movements after systemic or intrastriatal injection of the cholinergic agonist oxotremorine. Therefore, impairment of the cAMP pathway in association with an increased cholinergic tone creates alterations in striatal neuron functions that can promote the onset of dystonia. Our results also provide evidence that untreated and oxotremorine-treated Gnal-haplodeficient mice are powerful models with which to study presymptomic and symptomatic stages of DYT25-associated dystonia, respectively.

There are few data on leishmaniases and sandflies in Oman Sultanate. We carried out an eco-epidemiological study in 1998 in the two main mountains of the country, the Sharqiyah and the Dhofar. This study allowed us to isolate and identify three Leishmania strains from patients exhibiting cutaneous leishmaniasis. The typing carried out by isoenzymatic study and by molecular biology were congruent: two strains of Leishmania donovani zymodeme (Z) MON-31 isolated in the Sharqiyah and one L. tropica ZROM102 (ZMON-39 variant for 4 isoenzymes) from the Dhofar. No strain was isolated from canids. The study of sandflies identified 14 species distributed in the genera Phlebotomus, Sergentomyia and Grassomyia: Ph. papatasi, Ph. bergeroti, Ph. duboscqi, Ph. alexandri, Ph. saevus, Ph. sergenti, Se. fallax, Se. baghdadis, Se. cincta, Se. christophersi, Se. clydei, Se. tiberiadis, Se. africana, and Gr. dreyfussi. In Sharqiyah, the only candidate for the transmission of L. donovani was Ph. alexandri, but the low densities observed of this species do not argue in favor of any role. In Dhofar, Ph. sergenti is the most important proven vector of L. tropica, but Ph. saevus, a locally much more abundant species, constitutes a good candidate for transmission.Leishmanioses et phlébotomes au Sultanat d’Oman.Il existe peu de données sur les leishmanioses et les phlébotomes en Oman. Nous y avons mené en 1998 une étude éco-épidémiologique dans les deux principaux massifs montagneux du pays, la Sharqiyah et le Dhofar. Cette étude nous a permis d’isoler et d’identifier trois souches de Leishmania à partir de patients présentant des leishmanioses cutanées. Les typages menés par étude isoenzymatique et par biologie moléculaire ont été congruents : deux souches de Leishmania donovani ZMON-31 isolées dans la Sharqiyah et une de L. tropica ZROM102 (ZMON-39 variant pour 4 isoenzymes) originaire du Dhofar. Aucune souche n’a été isolée à partir de Canidés. L’étude des Phlébotomes a permis d’identifier 14 espèces réparties dans les genres Phlebotomus, Sergentomyia et Grassomyia : Ph. papatasi, Ph. bergeroti, Ph. duboscqi, Ph. alexandri, Ph. saevus, Ph. sergenti, Se. fallax, Se. baghdadis, Se. cincta, Se. christophersi, Se. clydei, Se. tiberiadis, Se. africana et Gr. dreyfussi. Dans la Sharqiyah, la seule espèce candidate à la transmission de L. donovani est Ph. alexandri mais les faibles densités observées de cette espèce ne plaident pas en faveur d’un quelconque rôle. Dans le Dhofar, Ph. sergenti est le principal vecteur prouvé de L. tropica mais Ph. saevus, espèce localement bien plus abondante, constitue une bonne espèce candidate à la transmission.

In advanced Parkinson's disease, l-DOPA treatment causes the appearance of abnormal involuntary movements or l-DOPA-induced dyskinesia (LID). LID results in part from l-DOPA-induced activation of extracellular signal-regulated kinase (ERK) in the dopamine-denervated striatum. Activated ERK triggers nuclear responses, including phosphorylation of mitogen- and stress-activated protein kinase 1 (MSK1) and histone H3, and transcription of genes such as FosB. To determine the role of MSK1, wild type and MSK1 knockout mice with unilateral 6-hydroxydopamine lesion in the dorsolateral striatum were chronically treated with l-DOPA. The absence of MSK1 had no effect on the lesion or l-DOPA-induced ERK activation, but reduced l-DOPA-induced phosphorylation of histone H3 and FosB accumulation in the dopamine-denervated striatum. MSK1 deficiency also prevented the increase in Gαolf, the stimulatory α subunit of G protein coupling striatal dopamine D1 receptor to adenylyl cyclase. However, the intensity of LID was similar in MSK1-deficient and wild type mice. In conclusion, l-DOPA-induced activation of MSK1 contributes to histone H3 phosphorylation, induction of FosB, and Gαolf up-regulation but appears not to be necessary for the development of LID.

The ribosomal protein S6 (rpS6) is a component of the small 40S ribosomal subunit, involved in multiple physiological functions. Here, we examined the effects produced by haloperidol, a typical antipsychotic drug, on the phosphorylation of rpS6 at Ser240/244 in the striatum, a brain region involved in neurodegenerative and neuropsychiatric disorders. We found that administration of haloperidol increased Ser240/244 phosphorylation in a subpopulation of GABA-ergic medium spiny neurons (MSNs), which preferentially express dopamine D2 receptors (D2Rs). This effect was abolished by rapamycin, an inhibitor of the mammalian target of rapamycin complex 1 (mTORC1), or by PF470867, a selective inhibitor of the p70 ribosomal S6 kinase 1 (S6K1). We also found that the effect of haloperidol on Ser240/244 phosphorylation was prevented by functional inactivation of dopamine- and cAMP-regulated phosphoprotein of 32 kDa (DARPP-32), an endogenous inhibitor of protein phosphatase-1 (PP-1). In line with this observation, incubation of striatal slices with okadaic acid and calyculin A, two inhibitors of PP-1, increased Ser240/244 phosphorylation. These results show that haloperidol promotes mTORC1- and S6K1-dependent phosphorylation of rpS6 at Ser240/244, in a subpopulation of striatal MSNs expressing D2Rs. They also indicate that this effect is exerted by suppressing dephosphorylation at Ser240/244, through PKA-dependent activation of DARPP-32 and inhibition of PP-1.

D2/D3 dopamine receptors (D2R/D3R) agonists regulate Akt, but their effects display a complex time-course. In addition, the respective roles of D2R and D3R are not defined and downstream targets remain poorly characterized, especially in vivo. These issues were addressed here for D3R. Systemic administration of quinelorane, a D2R/D3R agonist, transiently increased phosphorylation of Akt and GSK-3β in rat nucleus accumbens and dorsal striatum with maximal effects 10 min after injection. Akt activation was associated with phosphorylation of several effectors of the mammalian target of rapamycin complex 1 (mTORC1): p70S6 kinase, ribosomal protein-S6 (Ser240/244), and eukaryotic initiation factor-4E binding protein-1. The action of quinelorane was antagonized by a D2/D3R antagonist, raclopride, and the selective D3R antagonist S33084, inactive by themselves. Furthermore, no effect of quinerolane was seen in knock-out mice lacking D3R. In drd1a-EGFP transgenic mice, quinelorane activated Akt/GSK-3β in both neurons expressing and lacking D1 receptor. Thus, the stimulation of D3R transiently activates the Akt/GSK-3β pathway in the two populations of medium-size spiny neurons of the nucleus accumbens and dorsal striatum. This effect may contribute to the influence of D3R ligands on reward, cognition, and processes disrupted in schizophrenia, drug abuse, and Parkinson's disease.

International audience; The nodes of Ranvier are essential regions for action potential conduction in myelinated fibers. They are enriched in multimolecular complexes composed of voltage-gated Nav and Kv7 channels associated with cell adhesion molecules. Cytoskeletal proteins ankyrin-G (AnkG) and βIV-spectrin control the organization of these complexes and provide mechanical support to the plasma membrane. IQCJ-SCHIP1 is a cytoplasmic protein present in axon initial segments and nodes of Ranvier. It interacts with AnkG and is absent from nodes and axon initial segments of βIV-spectrin and AnkG mutant mice. Here, we show that IQCJ-SCHIP1 also interacts with βIV-spectrin and Kv7.2/3 channels and self-associates, suggesting a scaffolding role in organizing nodal proteins. IQCJ-SCHIP1 binding requires a βIV-spectrin-specific domain and Kv7 channel 1-5-10 calmodulin-binding motifs. We then investigate the role of IQCJ-SCHIP1 in vivo by studying peripheral myelinated fibers in Schip1 knock-out mutant mice. The major nodal proteins are normally enriched at nodes in these mice, indicating that IQCJ-SCHIP1 is not required for their nodal accumulation. However, morphometric and ultrastructural analyses show an altered shape of nodes similar to that observed in βIV-spectrin mutant mice, revealing that IQCJ-SCHIP1 contributes to nodal membrane-associated cytoskeleton organization, likely through its interactions with the AnkG/βIV-spectrin network. Our work reveals that IQCJ-SCHIP1 interacts with several major nodal proteins, and we suggest that it contributes to a higher organizational level of the AnkG/βIV-spectrin network critical for node integrity.

When considering inherited diseases that can be treated by gene transfer into hematopoietic stem cells (HSCs), there are only two in which the HSC and progenitor cell distribution inside the bone marrow and its microenvironment are exactly the same as in a healthy subject: metachromatic leukodystrophy (MLD) and adrenoleukodystrophy (ALD). In all other settings [X-linked severe combined immunodeficiency (X-SCID), adenosine deaminase deficiency, Wiskott-Aldrich syndrome, and β-hemoglobinopathies], the bone marrow content of the different stem and precursor cells and the cells' relationship with the stroma have very specific characteristics. These peculiarities can influence the cells' harvesting and behavior in culture, and the postgraft uptake and further behavior of the gene-modified hematopoietic/precursor cells. In the present mini-review, we shall briefly summarize these characteristics and outline the possible consequences and challenges.

International audience; The interaction of glutamate and dopamine in the striatum is heavily dependent on signaling pathways that converge on the regulatory protein DARPP-32. The efficacy of dopamine/D1 receptor/PKA signaling is regulated by DARPP-32 phosphorylated at Thr-34 (the PKA site), a process that inhibits protein phosphatase 1 (PP1) and potentiates PKA action. Activation of dopamine/D1 receptor/PKA signaling also leads to dephosphorylation of DARPP-32 at Ser-97 (the CK2 site), leading to localization of phospho-Thr-34 DARPP-32 in the nucleus where it also inhibits PP1. In this study the role of glutamate in the regulation of DARPP-32 phosphorylation at four major sites was further investigated. Experiments using striatal slices revealed that glutamate decreased the phosphorylation states of DARPP-32 at Ser-97 as well as Thr-34, Thr-75, and Ser-130 by activating NMDA or AMPA receptors in both direct and indirect pathway striatal neurons. The effect of glutamate in decreasing Ser-97 phosphorylation was mediated by activation of PP2A. In vitro phosphatase assays indicated that the PP2A/PR72 heterotrimer complex was likely responsible for glutamate/Ca2+-regulated dephosphorylation of DARPP-32 at Ser-97. As a consequence of Ser-97 dephosphorylation, glutamate induced the nuclear localization in cultured striatal neurons of dephospho-Thr-34/dephospho-Ser-97 DARPP-32. It also reduced PKA-dependent DARPP-32 signaling in slices and in vivo Taken together, the results suggest that by inducing dephosphorylation of DARPP-32 at Ser-97 and altering its cytonuclear distribution, glutamate may counteract dopamine/D1 receptor/PKA signaling at multiple cellular levels.

© The Author(s)., Here, we unravel the mechanism of action of the Ikaros family zinc finger protein Helios (He) during the development of striatal medium spiny neurons (MSNs). He regulates the second wave of striatal neurogenesis involved in the generation of striatopallidal neurons, which express dopamine 2 receptor and enkephalin. To exert this effect, He is expressed in neural progenitor cells (NPCs) keeping them in the G1/G0 phase of the cell cycle. Thus, a lack of He results in an increase of S-phase entry and S-phase length of NPCs, which in turn impairs striatal neurogenesis and produces an accumulation of the number of cycling NPCs in the germinal zone (GZ), which end up dying at postnatal stages. Therefore, He−/− mice show a reduction in the number of dorso-medial striatal MSNs in the adult that produces deficits in motor skills acquisition. In addition, overexpression of He in NPCs induces misexpression of DARPP-32 when transplanted in mouse striatum. These findings demonstrate that He is involved in the correct development of a subset of striatopallidal MSNs and reveal new cellular mechanisms for neuronal development., This study was supported by grants from the Ministerio de Economıa y ́ Competitividad (BFU2010-19630 to C.V.-A.; SAF 2014-57160-R, to J. A.; SAF2015- 66505-R to J.M.C.), and Instituto de Salud Carlos III-Subdirección General de Evaluación, and European Regional Development Fund (ERDF) [CIBERNED, to J. A and C.V.-A.; and RETICS (RD12/0019/0002; Red de Terapia Celular), to J.M.C.], Spain; Generalitat de Catalunya (2014SGR-968 to J.A.); Fundació la Marató de TV3 (20140130/1 to J.A.); and CHDI Foundation (A-7332 to J.M.C.). M.P. was a fellow from the Generalitat de Catalunya, Spain and E.C. was a fellow of the Ministerio de Economía y Competitividad, Spain

The structure and function of spines and excitatory synapses are under the dynamic control of multiple signalling networks. Although tyrosine phosphorylation is involved, its regulation and importance are not well understood. Here we study the role of Pyk2, a non-receptor calcium-dependent protein-tyrosine kinase highly expressed in the hippocampus. Hippocampal-related learning and CA1 long-term potentiation are severely impaired in Pyk2-deficient mice and are associated with alterations in NMDA receptors, PSD-95 and dendritic spines. In cultured hippocampal neurons, Pyk2 has autophosphorylation-dependent and -independent roles in determining PSD-95 enrichment and spines density. Pyk2 levels are decreased in the hippocampus of individuals with Huntington and in the R6/1 mouse model of the disease. Normalizing Pyk2 levels in the hippocampus of R6/1 mice rescues memory deficits, spines pathology and PSD-95 localization. Our results reveal a role for Pyk2 in spine structure and synaptic function, and suggest that its deficit contributes to Huntington’s disease cognitive impairments.

We describe a retinal endovascular fibrinolysis technique to directly reperfuse experimentally occluded retinal veins using a simple micropipette.Retinal vein occlusion was photochemically induced in 12 eyes of 12 minipigs: after intravenous injection of 10% fluorescein (1-mL bolus), the targeted retinal vein segment was exposed to thrombin (50 units) and to Argon laser (100-200 mW) through a pars plana approach. A beveled micropipette with a 30-μm-diameter sharp edge was used for micropuncture of the occluded vein and endovascular microinjection of tissue plasminogen activator (50 μg/mL) in 11 eyes. In one control eye, balanced salt solution was injected. The lesion site was examined histologically.Retinal vein occlusion was achieved in all cases. Endovascular microinjection of tissue plasminogen activator or balanced salt solution led to reperfusion of the occluded retinal vein in all cases. Indicative of successful reperfusion were the following: continuous endovascular flow, unaffected collateral circulation, no optic disk ischemia, and no venous wall bleeding. However, balanced salt solution injection was accompanied by thrombus formation at the punctured site, whereas no thrombus was observed with tissue plasminogen activator injection.Retinal endovascular fibrinolysis constitutes an efficient method of micropuncture and reperfusion of an experimentally occluded retinal vein. Thrombus formation at the punctured site can be prevented by injection of tissue plasminogen activator.

Objectives:An animal model of cochlear implantation has been developed, and the hearing threshold was evaluated after different surgical procedures. The effect of perioperative systemic administration of erythropoietin on the hearing loss induced by cochlear implantation was tested.Methods:Twenty-nine guinea pigs with normal hearing underwent implantation of a 254-μm-diameter array through a cochleostomy. The effects on hearing of cochleostomy and transient and long-term array implantation (21 days) were assessed by testing of the auditory brain stem responses and compound action potentials. Eleven implanted animals received intraperitoneal administration of erythropoietin. Selected computed tomographic scans and cochlear histologic studies were performed 1 month after implantation to confirm proper placement of the array. The erythropoietin concentration at the time of surgery was assessed in samples of perilymph, cerebrospinal fluid, and blood.Results:The cochleostomy and transient array insertion had no effect on hearing thresholds. Long-term array implantation induced a stable decrease of hearing threshold (30 dB), a decrease that was reduced by 12 dB in erythropoietin-treated animals. The erythropoietin-treated animals had better hearing preservation at higher frequencies. Fibrosis surrounding the array was seen in both groups.Conclusions:The hearing loss observed was probably due to the presence of the array in the cochlea. The intraperitoneal injection of erythropoietin improved the hearing threshold shift induced by implantation.

During human glomerulonephritis, the severity of injuries correlates with glomerular fibrin deposits, which are tightly regulated by the intraglomerular fibrinolytic system. Here, we evaluated the role of vitronectin (VTN; also known as complement S protein), the principal cofactor of the plasminogen activator inhibitor-1 (PAI-1), in a mouse model of acute glomerulonephritis. We found that in mice subjected to nephrotoxic serum, the absence of VTN resulted in a lower glomerular PAI-1 activity and a higher glomerular fibrinolytic activity. Challenged VTN(-/-) mice displayed significantly less fibrin deposits, proteinuria, and renal failure than their wild-type counterparts. Notably, this protective effect afforded by VTN deficiency was still observed after a C3 depletion. Finally, the injection of VTN(+/+) serum in VTN(-/-) mice induced the glomerular deposition of VTN, increased PAI-1 deposition, decreased glomerular fibrinolytic activity, and aggravated glomerular injury. As in mice, abundant glomerular VTN deposits were also observed in patients with severe glomerulonephritis. Here, we show that plasma-exchange therapy, admittedly beneficial in this clinical context, induces a significant depletion in circulating VTN, which might modulate PAI-1 activity locally and accelerate the clearance of fibrin deposits in the glomeruli. Collectively, these results demonstrate that VTN exerts a deleterious role independently from complement, by directing PAI-dependent fibrinolysis in the glomerular compartment.

Alcohol consumption is a moderately heritable trait, but the genetic basis in humans is largely unknown, despite its clinical and societal importance. We report a genome-wide association study meta-analysis of ∼2.5 million directly genotyped or imputed SNPs with alcohol consumption (gram per day per kilogram body weight) among 12 population-based samples of European ancestry, comprising 26,316 individuals, with replication genotyping in an additional 21,185 individuals. SNP rs6943555 in autism susceptibility candidate 2 gene ( AUTS2 ) was associated with alcohol consumption at genome-wide significance ( P = 4 × 10 −8 to P = 4 × 10 −9 ). We found a genotype-specific expression of AUTS2 in 96 human prefrontal cortex samples ( P = 0.026) and significant ( P < 0.017) differences in expression of AUTS2 in whole-brain extracts of mice selected for differences in voluntary alcohol consumption. Down-regulation of an AUTS2 homolog caused reduced alcohol sensitivity in Drosophila ( P < 0.001). Our finding of a regulator of alcohol consumption adds knowledge to our understanding of genetic mechanisms influencing alcohol drinking behavior.

Differentiating agents have been proposed to overcome the impaired cellular differentiation in acute myeloid leukemia (AML). However, only the combinations of all-trans retinoic acid or arsenic trioxide with chemotherapy have been successful, and only in treating acute promyelocytic leukemia (also called AML3). We show that iron homeostasis is an effective target in the treatment of AML. Iron chelating therapy induces the differentiation of leukemia blasts and normal bone marrow precursors into monocytes/macrophages in a manner involving modulation of reactive oxygen species expression and the activation of mitogen-activated protein kinases (MAPKs). 30% of the genes most strongly induced by iron deprivation are also targeted by vitamin D3 (VD), a well known differentiating agent. Iron chelating agents induce expression and phosphorylation of the VD receptor (VDR), and iron deprivation and VD act synergistically. VD magnifies activation of MAPK JNK and the induction of VDR target genes. When used to treat one AML patient refractory to chemotherapy, the combination of iron-chelating agents and VD resulted in reversal of pancytopenia and in blast differentiation. We propose that iron availability modulates myeloid cell commitment and that targeting this cellular differentiation pathway together with conventional differentiating agents provides new therapeutic modalities for AML.

Focal adhesion kinase (FAK) regulates numerous cellular functions and is critical for processes ranging from embryo development to cancer progression. Although autophosphorylation on Tyr-397 appears required for FAK functions in vitro, its role in vivo has not been established. We addressed this question using a mutant mouse (fakDelta) deleted of exon 15, which encodes Tyr-397. The resulting mutant protein FAKDelta is an active kinase expressed at normal levels. Our results demonstrate that the requirement for FAK autophosphorylation varies during development. FAK(Delta/Delta) embryos developed normally up to embryonic day (E) 12.5, contrasting with the lethality at E8.5 of FAK-null embryos. Thus, autophosphorylation on Tyr-397 is not required for FAK to achieve its functions until late mid-gestation. However, FAK(Delta/Delta) embryos displayed hemorrhages, edema, delayed artery formation, vascular remodeling defects, multiple organ abnormalities, and overall developmental retardation at E13.5-14.5, and died thereafter demonstrating that FAK autophosphorylation is also necessary for normal development. Fibroblasts derived from mutant embryos had a normal stellate morphology and expression of focal adhesion proteins, Src family members, p53, and Pyk2. In contrast, in FAK(Delta/Delta) fibroblasts and endothelial cells, spreading and lamellipodia formation were altered with an increased size and number of focal adhesions, enriched in FAKDelta. FAK mutation also decreased fibroblast proliferation. These results show that the physiological functions of FAK in vivo are achieved through both autophosphorylation-independent and autophosphorylation-dependent mechanisms.

A single exposure to psychostimulants or morphine is sufficient to induce persistent locomotor sensitization, as well as neurochemical and electrophysiological changes in rodents. Although it provides a unique model to study the bases of long-term behavioral plasticity, sensitization mechanisms remain poorly understood. We investigated in the mouse, a species suited for transgenic studies, the mechanisms of locomotor sensitization showed by the increased response to a second injection of drug (two-injection protocol of sensitization, TIPS). The first cocaine injection induced a locomotor sensitization that was completely context-dependent, increased during the first week, and persisted 3 months later. The induction of sensitized responses to cocaine required dopamine D1 and glutamate NMDA receptors. A single injection of the selective dopamine transporter blocker GBR12783 was sufficient to activate extracellular signal-regulated kinase (ERK) in the striatum to the same level as cocaine and to induce sensitization to cocaine, but not to itself. The induction of sensitization was sensitive to protein synthesis inhibition by anisomycin after cocaine administration. Morphine induced a pronounced context-dependent sensitization that crossed with cocaine. Sensitization to morphine injection was prevented in knockin mutant mice bearing a Thr-34-Ala mutation of DARPP-32, which suppresses its ability to inhibit protein phosphatase-1 (PP1), but not mutation of Thr-75 or Ser-130. These results combined with previous ones show that TIPS in mouse is a context-dependent response, which involves an increase in extracellular dopamine, stimulation of D1 and NMDA receptors, regulation of the cAMP-dependent and ERK pathways, inhibition of PP1, and protein synthesis. It provides a simple and sensitive paradigm to study the mechanisms of long-term effects of drugs of abuse.

International audience; Stem cells generate self-renewing and differentiating progeny over many rounds of asymmetric divisions. How stem cell growth rate and size are maintained over time remains unknown. We isolated mutations in a Drosophila melanogaster gene, wicked (wcd), which induce premature differentiation of germline stem cells (GSCs). Wcd is a member of the U3 snoRNP complex required for pre-ribosomal RNA maturation. This general function of Wcd contrasts with its specific requirement for GSC self-renewal. However, live imaging of GSCs within their niche revealed a pool of Wcd-forming particles that segregate asymmetrically into the GSCs on mitosis, independently of the Dpp signal sent by the niche. A fraction of Wcd also segregated asymmetrically in dividing larval neural stem cells (NSCs). In the absence of Wcd, NSCs became smaller and produced fewer neurons. Our results show that regulation of ribosome synthesis is a crucial parameter for stem cell maintenance and function.

International audience; Drugs of abuse induce neuroadaptations through regulation of gene expression. Although much attention has focused on transcription factor activities, new concepts have recently emerged on the role of chromatin remodelling as a prerequisite for regulation of gene expression in neurons. Thus, for transcription to occur, chromatin must be decondensed, a dynamic process that depends on post-translational modifications of histones. We review here these modifications with a particular emphasis on the role of histone H3 phosphorylation at the promoter of specific genes, including c-fos and c-jun. We trace the signalling pathways involved in H3 phosphorylation and provide evidence for a role of mitogen and stress-activated protein kinase-1 (MSK1) downstream from the MAPK/extracellular-signal regulated kinase (ERK) cascade. In response to cocaine, MSK1 controls an early phase of histone H3 phosphorylation at the c-fos promoter in striatal neurons. MSK1 action may be potentiated by the concomitant inhibition of protein phosphatase 1 by nuclear translocation of dopamine- and cAMP-regulated phosphoprotein Mr = 32 000. H3 phosphorylation by MSK1 is critically involved in c-fos transcription, and cocaine-induced locomotor sensitization. Thus, ERK plays a dual role in gene regulation and drug addiction by direct activation of transcription factors and by chromatin remodelling.

Exposure to cues previously associated with intake of substances of abuse can promote drug related responses. In this study, we have examined the effect of exposure to a drug-associated context on the expression of morphine psychomotor sensitization. We show that sensitization is markedly increased in mice examined 4 weeks after the last morphine injection. In addition, this incubation period confers to the environment paired with morphine the ability to increase ERK phosphorylation in the shell (but not the core) of the nucleus accumbens. Using transgenic mice with enhanced green fluorescent protein (EGFP) expression under the control of the dopamine D1 receptor (D1R) (Drd1a-EGFP) or D2 receptor promoter (Drd2-EGFP) we show that context-dependent ERK phosphorylation is restricted to D1R-expressing medium spiny neurons. Furthermore, this effect depends on D1R activation. These data show that, following repeated morphine injections, a drug-free period induces context-dependent phosphorylation of ERK in a specific population of neurons within the nucleus accumbens shell. This activation is associated to enhanced psychomotor sensitization and may be implicated in context-elicited drug seeking induced by repeated exposure to drugs of abuse.